Mutant pores

ABSTRACT

The invention relates to mutant forms of CsgG. The invention also related to analyte detection and characterisation using CsgG.

This application is a continuation of U.S. application Ser. No.15/507,947, filed on Mar. 1, 2017, which is a national stage filingunder 35 U.S.C. § 371 of PCT International Application No.PCT/EP2015/069965, which has an international filing date of Sep. 1,2015, and claims foreign priority benefits under 35 U.S.C. § 119(a)-(d)or 35 U.S.C. § 365(b) of British application number 1415455.3, filedSep. 1, 2014, British application number 1422079.2, filed Dec. 11, 2014,British application number 1506489.2, filed Apr. 16, 2015, Britishapplication number 1506754.9, filed Apr. 21, 2015, British applicationnumber 1508287.8, filed May 14, 2015, British application number1511203.0, filed Jun. 25, 2015, and British application number1515240.8, filed Aug. 27, 2015. The contents of the aforementionedapplications are herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to novel protein pores and their uses. Inparticular it relates to biological nanopores in nucleic acid sequencingapplications, and molecular sensing.

The invention relates to mutant forms of CsgG. The invention alsorelates to analyte detection and characterisation using CsgG.

BACKGROUND OF THE INVENTION

Protein pores are membrane spanning polypeptides and complexes that forma channel in the membrane through which ions and certain molecules maypass. The minimum diameter of the channel is typically in the nanometre(10-⁹ metre) range hence giving certain of these polypeptides the name“nanopores”.

Nanopores have great potential as biological sensors. When an electricalpotential is applied across a membrane-bound nanopore, ions flow throughthe channel. This flow of ions can be measured as an electrical current.Suitable electrical measurement techniques using single channelrecording equipment are described in, for example, WO 2000/28312 and D.Stoddart et al., Proc. Natl. Acad. Sci., 2010, 10 7702-7. Multi-channelrecording techniques are described, for example, in WO 2009/077734.

A molecule translating though the pore, or binding in or near the poreacts to obstruct and thereby reduce the ion flow through the channel.The degree of reduction in ion flow, as measured by the reduction inelectrical current, is indicative of the size of the obstruction within,or in the vicinity of, the pore. The measured electrical current cantherefore be used as a measure of the size or degree of obstruction tothe channel. The changes in electrical current can be used to identifythat a molecule, or part of a molecule, has bound at or near the pore(molecular sensing), or in certain systems, it can be used to determinethe identity of a molecule that is present within the pore based on itssize (nucleic acid sequencing).

The “Strand Sequencing” method is known for sequencing nucleic acidsusing biological nanopores. On passing a single polynucleotide strandthrough a nanopore, the bases on individual nucleotides are determinedby the changes in measured electrical current as they pass transientlythrough the channel of the nanopore. This method offers significant timeand cost savings over historic methods of nucleic acid sequencing.

Previously reported protein nanopores, such as the mutant MspA (Manraoet al., Nature Biotechnology, 2012, 30(4), 349-353) and alpha-hemolysinnanopores (Nat. Nanotechnol., 2009, 4(4), 265-70) have been used fornucleic acid sequencing using the “Strand Sequencing” approach.Similarly, for protein 40 sensing other pores such as alpha-hemolysin (JAm Chem Soc., 2012, 134(5), 2781-7) and ClyA (Am. Chem. Soc. Nano. 2014,8(12), 12826-35) (J. Am. Chem. Soc., 2013, 135(36), 13456-63) have alsobeen adapted.

There remains a need for new nanopores that overcome the deficiencies ofthe prior art, not least in optimising the dimensions andcharacteristics of the pore for molecular sensing applications, and forexample, nucleic acid sequencing applications.

Nanopore sensing is an approach to sensing that relies on theobservation of individual binding or interaction events between analytemolecules and a receptor. Nanopore sensors can be created by placing asingle pore of nanometer dimensions in an insulating membrane andmeasuring voltage-driven ionic transport through the pore in thepresence of analyte molecules. The identity of an analyte is revealedthrough its distinctive current signature, notably the duration andextent of current block and the variance of current levels.

There is currently a need for rapid and cheap nucleic acid (e.g. DNA orRNA) sequencing technologies across a wide range of applications.Existing technologies are slow and expensive mainly because they rely onamplification techniques to produce large volumes of nucleic acid andrequire a high quantity of specialist fluorescent chemicals for signaldetection. Nanopore sensing has the potential to provide rapid and cheapnucleic acid sequencing by reducing the quantity of nucleotide andreagents required.

Two of the essential components of sequencing nucleic acids usingnanopore sensing are (1) the control of nucleic acid movement throughthe pore and (2) the discrimination of nucleotides as the nucleic acidpolymer is moved through the pore. In the past, to achieve nucleotidediscrimination the nucleic acid has been passed through a mutant ofhemolysin. This has provided current signatures that have been shown tobe sequence dependent. It has also been shown that a large number ofnucleotides contribute to the observed current when a hemolysin pore isused, making a direct relationship between observed current andpolynucleotide challenging.

While the current range for nucleotide discrimination has been improvedthrough mutation of the hemolysin pore, a sequencing system would havehigher performance if the current differences between nucleotides couldbe improved further. In addition, it has been observed that when thenucleic acids are moved through a pore, some current states show highvariance. It has also been shown that some mutant hemolysin poresexhibit higher variance than others. While the variance of these statesmay contain sequence specific information, it is desirable to producepores that have low variance to simplify the system. It is alsodesirable to reduce the number of nucleotides that contribute to theobserved current.

SUMMARY OF THE INVENTION

The inventors have identified the structure of the bacterial amyloidsecretion channel CsgG. The CsgG channel is a trans-membrane oligomericprotein that forms a channel with a minimum diameter of approximately0.9 nm. The structure of the CsgG nanopore renders it suitable for usein protein sensing applications, in particular in nucleic acidsequencing. Modified versions of the CsgG polypeptide may serve tofurther enhance the suitability of the channel for such particularapplications.

The CsgG pore offers an advantage over existing protein pores such asClyA or alpha-hemolysin in that the structure is favourable for DNAsequencing applications. The CsgG pore has a more favourable aspectratio comprising a shorter trans-membrane channel than ClyA. The CsgGpore also has a wider channel opening compared to the alpha-hemolysinpore. This can facilitate the attachment of enzymes for certainapplications, for example nucleic acid sequencing applications. In theseembodiments, it can also minimize the length of the nucleic acid strandsection positioned between the enzyme and the reading head (defined asthe narrowest pore section) leading to an improved read-out signal. Thenarrow inner constriction of the channel of the CsgG pore alsofacilitates the translocation of single stranded DNA in embodiments ofthe invention involving nucleic acid sequencing. The constriction iscomposed of two annular rings formed by juxtaposition of tyrosineresidues at position 51 (Tyr 51) in the adjacent protein monomers, andalso the phenylalanine and asparagine residues at positions 56 and 55respectively (Phe 56 and Asn 55). The dimensions of the constriction canbe modified. ClyA has a much wider inner constriction which allows thepassage of double stranded DNA which is currently not used forsequencing. The alpha-hemolysin pore has one 1.3 nm-wide innerconstriction but also a 2 nm-wide beta barrel which features additionalreading heads.

In a first aspect, the invention relates to a method for molecularsensing comprising:

-   -   a) providing a CsgG biological pore formed of at least one CsgG        monomer within an insulating layer;    -   b) applying an electrical potential across the insulating layer        thereby establishing flow of electrical current through the        biological pore;    -   c) contacting the CsgG biological pore with a test substrate;        and    -   d) measuring the electrical current flow through the biological        pore.

Typically, the insulating layer is a membrane, such as a lipid bilayer.In an embodiment, the electrical current through the pore is carried bya flow of soluble ions from a first side of the insulating layer to thesecond side of the insulating layer.

In an embodiment of the invention, the molecular sensing is analytedetection. In a specific embodiment, the method for analyte detectioncomprises after step (d) the further step of determining the presence ofthe test substrate by a reduction in electrical current through thebiological pore compared to the electrical current through thebiological pore when the test substrate is absent.

In an alternative embodiment of the invention, the molecular sensing isnucleic acid sequencing. Typically, the type of nucleic acid sequencedby said method is DNA or RNA. In specific embodiments of the invention,the CsgG biological pore is adapted to accommodate additional accessoryproteins. Typically, the additional accessory proteins are nucleicacid-processing enzymes selected from the group consisting of: DNA orRNA polymerases; isomerases; topoisomerases; gyrases; telomerases;exonucleases; and helicases.

In embodiments of the invention, the CsgG biological pore is a modifiedCsgG pore, wherein the modified CsgG pore has at least one modificationto the monomeric wild-type E-coli CsgG polypeptide sequence in at leastone of the CsgG monomers forming the CsgG pore. Typically, the samemodification is made to all the CsgG monomers forming the CsgG pore. Inspecific embodiments of the invention, the modified CsgG monomer has apolypeptide sequence from positions 38 to 63 according to SEQ ID NOs 4to 388.

In a second aspect, the invention relates to modified CsgG biologicalpore comprising at least one CsgG monomer, wherein the modified CsgGbiological pore has no more than one channel constriction with adiameter in the range from 0.5 nm to 1.5 nm. Typically, the modificationis between positions 38 to 63 of the CsgG monomeric polypeptidesequence. Suitably, the modification is at a position selected from:Tyr51; Asn55; and Phe 56. In specific embodiments, the modification isat position Tyr 51, or at both of positions Asn55 and Phe56.

In embodiments of the invention, the modification to the CsgG monomer isselected from the group consisting of substitution of the naturallyoccurring amino acid; deletion of the naturally occurring amino acid;and modification of the naturally-occurring amino acid side chain.Suitably, the modification reduces or removes the steric encumbrance ofthe unmodified amino acid. In specific embodiments, at least one CsgGmonomer of the pore has a polypeptide sequence from positions 38 to 63according to SEQ ID NOs 4 to 388.

In a third aspect, the invention relates to the isolated polypeptideencoding the at least one CsgG monomer of the modified CsgG biologicalpore of the second aspect of the invention.

In a fourth aspect, the invention relates to isolated nucleic acidsencoding the isolated polypeptides of the third aspect of the invention.

In a fifth aspect, the invention relates to a biosensor comprising:

-   -   a) An insulating layer;    -   b) A CsgG biological pore within the insulating layer; and    -   c) Apparatus for measuring an electrical current through the        biological pore.

In specific embodiments, the CsgG biological pore in the biosensor is amodified CsgG biological pore according to the second aspect of theinvention.

In a sixth aspect, the invention relates to the use of a CsgG biologicalpore for biological sensing applications, wherein the biological sensingapplication is analyte detection or nucleic acid sequencing.

In an embodiment of the sixth aspect of the invention, the nucleic acidsequencing is DNA sequencing or RNA sequencing.

The inventors have surprisingly demonstrated that CsgG and novel mutantsthereof may be used to characterise analytes, such as polynucleotides.The invention concerns mutant CsgG monomers in which one or moremodifications have been made to improve the ability of the monomer tointeract with an analyte, such as a polynucleotide. The inventors havealso surprisingly demonstrated that pores comprising the novel mutantmonomers have an enhanced ability to interact with analytes, such aspolynucleotides, and therefore display improved properties forestimating the characteristics of analytes, such as the sequence ofpolynucleotides. The mutant pores surprisingly display improvednucleotide discrimination. In particular, the mutant pores surprisinglydisplay an increased current range, which makes it easier todiscriminate between different nucleotides, and a reduced variance ofstates, which increases the signal-to-noise ratio. In addition, thenumber of nucleotides contributing to the current as the polynucleotidemoves through the pore is decreased. This makes it easier to identify adirect relationship between the observed current as the polynucleotidemoves through the pore and the polynucleotide. In addition, the mutantpores may display an increased throughput, i.e. are more likely tointeract with an analyte, such as a polynucleotide. This makes it easierto characterise analytes using the pores. The mutant pores may insertinto a membrane more easily.

Accordingly, the invention provides a mutant CsgG monomer comprising avariant of the sequence shown in SEQ ID NO: 390, wherein the variantcomprises a mutation at one or more of positions Y51, N55 and F56.

Accordingly, the invention provides a mutant CsgG monomer comprising avariant of the sequence shown in SEQ ID NO: 390, wherein the variantcomprises one or more of the following: (i) one or more mutations at thefollowing positions (i.e. mutations at one or more of the followingpositions) N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142,T150 and R192; (ii) mutations at Y51/N55, Y51/F56, N55/F56 orY51/N55/F56; (iii) Q42R or Q42K; (iv) K49R; (v) N102R, N102F, N102Y orN102W; (vi) D149N, D149Q or D149R; (vii) E185N, E185Q or E185R; (viii)D195N, D195Q or D195R; (ix) E201N, E201Q or E201R; (x) E203N, E203Q orE203R; and (xi) deletion of one or more of the following positions F48,K49, P50, Y51, P52, A53, S54, N55, F56 and S57.

The invention also provides:

-   -   a construct comprising two or more covalently attached CsgG        monomers, wherein at least one of the monomers is a mutant        monomer of the invention;    -   a polynucleotide which encodes a mutant monomer of the invention        or a construct of the invention;    -   a homo-oligomeric pore derived from CsgG comprising identical        mutant monomers of the invention or identical constructs of the        invention;    -   a hetero-oligomeric pore derived from CsgG comprising at least        one mutant monomer of the invention or at least one construct of        the invention;    -   a method for determining the presence, absence or one or more        characteristics of a target analyte, comprising:        -   a) contacting the target analyte with a CsgG pore or a            mutant thereof such that the target analyte moves with            respect to the pore; and        -   b) taking one or more measurements as the analyte moves with            respect to the pore and thereby determining the presence,            absence or one or more characteristics of the analyte;    -   a method of forming a sensor for characterising a target        polynucleotide, comprising forming a complex between a CsgG pore        or a mutant thereof and a polynucleotide binding protein and        thereby forming a sensor for characterising the target        polynucleotide;    -   a sensor for characterising a target polynucleotide, comprising        a complex between a CsgG pore or a mutant thereof and a        polynucleotide binding protein;    -   use of a CsgG pore or a mutant thereof to determine the        presence, absence or one or more characteristics of a target        analyte;    -   a kit for characterising a target analyte comprising (a) a CsgG        pore or a mutant thereof and (b) the components of a membrane;    -   an apparatus for characterising target analytes in a sample,        comprising (a) a plurality of a CsgG pores or mutants thereof        and (b) a plurality of membranes;    -   a method of characterising a target polynucleotide, comprising:        -   a) contacting the polynucleotide with a CsgG pore or a            mutant thereof, a polymerase and labelled nucleotides such            that phosphate labelled species are sequentially added to            the target polynucleotide by the polymerase, wherein the            phosphate species contain a label specific for each            nucleotide; and        -   b) detecting the phosphate labelled species using the pore            and thereby characterising the polynucleotide; and    -   a method of producing a mutant monomer of the invention or a        construct of the invention, comprising expressing a        polynucleotide of the invention in a suitable host cell and        thereby producing a mutant monomer of the invention or a        construct.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a side cross-sectional view of the structure of a CsgGnonamer in its channel conformation in ribbon and surfacerepresentation.

FIG. 2 shows a cross-section of CsgG channel constriction (i.e. the porereading head in the context of nanopore sensing applications) andrelevant diameter measurements.

FIG. 3 shows the structural motif that contributes to the poreconstriction comprising three stacked concentric side-chain layers: Tyr51, Asn 55 and Phe 56.

FIG. 4 shows the sequence homology in CsgG homologues, including themultiple sequence alignment of CsgG-like proteins (SEQ ID NO: 442 to SEQID NO: 448). The selected sequences were chosen from monophyletic cladesacross the phylogenetic tree of CsgG-like sequences (not shown), to givea representative view of sequence diversity. Secondary structureelements are shown as arrows or bars for β-strands and α-helices,respectively, and are based on the E. coli CsgG crystal structure.Importantly, the residues equivalent to E. coli Tyr 51, Asn55 and Phe 56are highlighted by arrows. These residues form the pore's innerconstriction, i.e. the pore reading head in the context of nanoporesensing applications.

FIG. 5 shows representative single-channel current recordings (a) andconductance histogram (b) of CsgG reconstituted in planar phospholipidbilayers and measured under an electrical field of +50 mV (n=33) or −50mV (n=13).

FIG. 6 shows single-channel current recordings of PPB-reconstituted CsgGat +50 mV or −50 mV and supplemented with incremental concentrations ofCsgE. Horizontal scale bars lie at 0 pA.

FIG. 7 shows a, Raw negative-stain EM image of C8E4/LDAO-solubilizedCsgG. Arrows indicate the different particle populations as labelled inthe size exclusion profile shown in g, being (I) aggregates of CsgGnonamers, (II) CsgG octadecamers and (III) CsgG nonamers. Scale bar, 20nm. b, Representative class average for top and side views of theindicated oligomeric states. c, Rotational autocorrelation functiongraph of LDAO-solubilized CsgG in top view, showing nine-fold symmetry.d, Raw negative-stain EM image of CsgG_(C1S). Arrows indicate thehexadecameric (IV) and octameric (V) particles observed bysize-exclusion chromatography in g. e, Representative class average forside views of CsgG_(C1S) oligomers. No top views were observed for thisconstruct. f, Table of elution volumes (EV) of CsgG_(C1S) and CsgGparticles observed by size-exclusion chromatography shown in g,calculated molecular mass (MW_(calc)), expected molecular mass(MW_(CsgG)) corresponding CsgG oligomerization state (CsgG_(n)) and theparticles' symmetry as observed by negative-stain EM and X-raycrystallography. g, Size-exclusion chromatogram of CsgG_(C1S) (black)and C8E4/LDAO-solubilized CsgG (grey) run on Superdex 200 10/300 GL (GEHealthcare). h, i, Ribbon representation of crystallized oligomers intop and side view, showing the D₈ hexadecamers for CsgG_(C1S) (h) and D₉octadecamers for membrane-extracted CsgG (i). One protomer is colouredin rainbow from N terminus (blue) to C terminus (red). The two C₈octamers (CsgG_(C1S)) or C₉ nonamers (CsgG) that form the tail-to-talldimers captured in the crystals are coloured blue and tan. r and θ giveradius and interprotomer rotation, respectively.

FIG. 8 shows an electron density map at 2.8 Å for CsgG_(C1S) calculatedusing NCS-averaged and density-modified experimental SAD phases, andcontoured at 1.5 g. The map shows the region of the channel construction(CL; a single protomer is labelled) and is overlaid on the final refinedmodel. CsgG_(C1S) is a mutant CsgG where the N-terminal Cys of themature CsgG sequence, i.e. Cys 1, is replaced by Ser, resulting in lackof lipid modification by the E. coli LOL pathway. This results in asoluble homooctameric oligomer that is present in a pre-poreconformation (see FIG. 42) contrary to the membrane-targetedhomononameric pore formed by native, lipid-modified CsgG (FIG. 43).

FIG. 9 shows top (FIG. 9a ) and side (FIG. 9b ) views of the CsgGconstriction modeled with a polyalanine chain threaded through thechannel in an extended conformation, shown in a C-terminal to N-terminaldirection. The modelled solvation of the polyalanine chain, position asin FIG. 9b , is shown in FIG. 9c with C-loops removed for clarity (shownsolvent molecules are those within 10 Å of the full polyalanine chain).

FIG. 10: Illustrates CsgG from E. coli.

FIG. 11: Illustrates the dimensions of CsgG.

FIG. 12: Illustrates single G translocation at 10 Å/ns. There is a largebarrier for entry of guanine into F56 ring in CsgG-Eco. *=G enters F56ring. A=G stops interacting with 56 ring. B=G stops interacting with 55ring. C=G stops interacting with 51 ring.

FIG. 13: Illustrates ssDNA translocation at 100 Å/ns. A larger force isrequired to pull the DNA through the constriction for CsgG-Eco.

FIG. 14: Illustrates ss DNA translocation at 10 Å/ns. CsgG-F56A-N55S andCsG-F56A-N55S-Y51A mutants both have a lower barrier for ssDNAtranslocation.

FIGS. 15 to 17: Mutant pores showing increased range compared withwild-type (WT).

FIGS. 18 and 19: Mutant pores showing increased throughput compared withwild-type (WT).

FIGS. 20 and 21: Mutant pore showing increased insertion compared withwild-type (WT).

FIG. 22: shows the DNA construct X used in Example 18. The regionlabelled 1 corresponded to 30 SpC3 spacers. The region labelled 2corresponded to SEQ ID NO: 415. The region labeled 3 corresponded tofour iSp18 spacers. The region labelled 4 corresponded to SEQ ID NO:416. The section labelled 5 corresponded to four 5-nitroindoles. Theregion labelled 6 corresponded to SEQ ID NO: 417. The region labelled 7corresponded to SEQ ID NO: 418. The region labelled 8 corresponded toSEQ ID NO: 419 which had four iSp18 spacers (the region labelled 9)attached at the 3′ end of SEQ ID NO: 419. At the opposite end of theiSp18 spacers was a 3′ cholesterol tether (labelled 10). The regionlabeled 11 corresponded to four SpC3 spacers.

FIG. 23: shows an example chromatography trace of Strep trap (GEHealthcare) purification of CsgG protein (x-axis label=elution volume(mL), Y-axis label=Absorbance (mAu)). The sample was loaded in 25 mMTris, 150 mM NaCl, 2 mM EDTA, 0.01% DDM and eluted with 10 mMdesthiobiotin. The elution peak in which CsgG protein eluted is labeledas E1.

FIG. 24: shows an example of a typical SDS-PAGE visualisation of CsgGprotein after the initial strep purification. A 4-20% TGX Gel (Bio Rad)was run at 300 V for 22 minutes in 1×TGS buffer. The gel was stainedwith Sypro Ruby stain. Lanes 1-3 show the main elution peak (labelled E1in FIG. 23) which contained CsgG protein as indicated by the arrow.Lanes 4-6 corresponded to elution fractions of the tail of the mainelution peak (labelled E1 in FIG. 23) which contained contaminants. Mshows the molecular weight marker used which was a Novex Sharp Unstained(unit=kD).

FIG. 25: Shows an example of a size exclusion chromatogram (SEC) of CsgGprotein (120 mL S200 GE healthcare, x-axis label=elution volume (mL),y-axis label=absorbance (mAu)). The SEC was carried out after streppurification and heating the protein sample. The running buffer for SECwas 25 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.01% DDM, 0.1% SDS, pH 8.0 andthe column was run at 1 mL/minute rate. The trace labelled X showsabsorbance at 220 nm and the trace labeled Y shows absorbance at 280 nm.The peak labelled with a star was collected.

FIG. 26: shows an example of a typical SDS-PAGE visualisation of CsgGprotein after SEC. A 4-20% TGX Gel (Bio Rad) was run at 300V for 22minutes in 1×TGS buffer and the gel was stained with Sypro Ruby stain.Lane 1 shows CsgG protein sample after strep purification and heatingbut before SEC. Lanes 2-8 show fractions collected across the peakrunning approximately 48 mL-60 mL of FIG. 25 (mid peak=55 mL) andlabelled with a star in FIG. 25. M shows the molecular weight markerused which was a Novex Sharp Unstained (unit=kD). The bar correspondingto the CsgG-Eco pore is indicated by an arrow.

FIGS. 27 to 33: Mutant pores showing increased range compared withwild-type (WT).

FIGS. 34 to 39: Mutant pores showing increased range compared withwild-type (WT).

FIG. 40 shows snap shots of the enzyme (T4 Dda-(E94C/C109A/C136A/A360C)(SEQ ID NO: 412 with mutations E94C/C109A/C136A/A360C and then(ΔM1)G1G2)) on top of the pore (CsgG-Eco-(Y51T/F56Q)-StrepII(C))9 (SEQID NO: 390 with mutations Y51T/F56Q where StepII(C) is SEQ ID NO: 435and is attached at the C-terminus pore mutant No. 20)) taken at 0 and 20ns during the simulations (Runs 1 to 3).

FIG. 41 shows snap shots of the enzyme (T4 Dda-(E94C/C109A/C136A/A360C)(SEQ ID NO: 412 with mutations E94C/C109A/C136A/A360C and then(ΔM1)G1G2)) on top of the pore (CsgG-Eco-(Y51T/F56Q)-StrepII(C))9 (SEQID NO: 390 with mutations Y51T/F56Q where StepII(C) is SEQ ID NO: 435and is attached at the C-terminus pore mutant No. 20)) taken at 30 and40 ns during the simulations (Runs 1 to 3).

FIG. 42 shows X-ray structure of CsgG_(C1S) in pre-pore conformation. a,Ribbon diagram of the CsgG_(C1S) monomer coloured as a blue to redrainbow from N terminus to C terminus. Secondary structure elements arelabelled according to the ABD-like fold, with the additional N-terminaland C-terminal a-helices and the extended loop connecting β1 and α1labelled αN, αC and C-loop (CL), respectively. b, Side view of theCsgG_(C1S) C8 octamer with subunits differentiated by colour and onesubunit oriented and coloured as in a.

FIG. 43 shows the structure of CsgG in its channel conformation. a,Amide I region (1,700-1,600 cm⁻¹) of ATR-FTIR spectra of CsgGC1S (blue)and membrane extracted CsgG (red). b, TM1 and TM2 sequence (SEQ ID NO:449 and SEQ ID NO: 450) (bilayer-facing residues in blue) and Congo redbinding of E. coli BW25141ΔcsgG complemented with wild-type csgG (WT),empty vector or csgG lacking the underlined fragments of TM1 or TM2.Data are representative of three biological replicates. c, Overlay ofCsgG monomer in pre-pore (light blue; TM1 pink, TM2 purple) and channelconformation (tan; TM1 green, TM2 orange). CL, C-loop. d, e, Side view(d) and cross-sectional view (e) of CsgG nonamers in ribbon and surfacerepresentation; helix 2, the core domain and TM hairpins are shown inblue, light blue and tan, respectively. A single protomer is coloured asin FIG. 42a . Magenta spheres show the position of Leu 2. OM, outermembrane.

FIG. 44 shows the CsgG channel constriction. a, Cross-section of CsgGchannel constriction and its solvent-excluded diameters. b, Theconstriction is composed of three stacked concentric side-chain layers:Tyr 51, Asn 55 and Phe 56, preceded by Phe 48 from the periplasmic side.c, CsgG channel topology. d, Congo red binding of E. coli BW25141ΔcsgGcomplemented with csgG (WT), empty vector or csgG carrying indicatedconstrictions mutants. Data are representative of six biologicalreplicates. e, f, Representative single channel current recordings (e)and conductance histogram (f) of CsgG reconstituted in planarphospholipid bilayers and measured under an electrical field of +50 mV(n=33) or −50 mV (n=13).

FIG. 45 shows a model of CsgG transport mechanism. a, NativePAGE of CsgE(E), CsgG (G) and CsgG supplemented with excess CsgE (E+G), showing theformation of a CsgG-CsgE complex (E−G*). Data are representative ofseven experiments, encompassing four protein batches. b, SDS-PAGE ofCsgE (E), CsgG (G) and the E−G* complex recovered from native PAGE. Dataare representative of two repetitions. M, molecular mass markers. c,Selected class averages of CsgG-CsgE particles. From left to right: topand side view visualized by cryo-EM, and comparison of negativelystained side views with CsgG nonamers. d, Cryo-EM averages of top andtilted side-viewed CsgE particles. Rotational autocorrelation showsnine-fold symmetry. e, Three-dimensional reconstruction of CsgG-CsgE(24A° resolution, 1,221 single particles) shows a nonameric particlecomprising CsgG (blue) and an additional density assigned as a CsgEnonamer (orange). f, Single-channel current recordings of PPBreconstituted CsgG at +50 mV or −50 mV and supplemented with incrementalconcentrations of CsgE. Horizontal scale bars lie at 0 pA. g, Tentativemodel for CsgG-mediated protein secretion. CsgG and CsgE are proposed toform a secretion complex that entraps CsgA (discussed in FIG. 54),generating an entropic potential over the channel. After capture of CsgAin the channel constriction, a DS-rectified Brownian diffusionfacilitates the progressive translocation of the polypeptide across theouter membrane.

FIG. 46 shows the Curli biosynthesis pathway in E. coli. The major curlisubunit CsgA (light green) is secreted from the cell as a solublemonomeric protein. The minor curli subunit CsgB (dark green) isassociated with the outer membrane (OM) and acts as a nucleator for theconversion of CsgA from a soluble protein to amyloid deposit. CsgG(orange) assembles into an oligomeric curli-specific translocationchannel in the outer membrane. CsgE (purple) and CsgF (light blue) formsoluble accessory proteins required for productive CsgA and CsgBtransport and deposition. CsgC forms a putative oxidoreductase ofunknown function. All curli proteins have putative Sec signal sequencesfor transport across the cytoplasmic (inner) membrane (IM).

FIG. 47 shows the in-solution oligomerization states of CsgG andCsgG_(C1S) analysed by size-exclusion chromatography and negative-stainelectron microscopy. a, Raw negative-stain EM image of C8E4/LDAOsolubilized CsgG. Arrows indicate the different particle populations aslabeled in the size exclusion profile shown in g, being (I) aggregatesof CsgG nonamers, (II) CsgG octadecamers and (III) CsgG nonamers. Scalebar, 20 nm. b, Representative class average for top and side views ofthe indicated oligomeric states. c, Rotational autocorrelation functiongraph of LDAO solubilized CsgG in top view, showing nine-fold symmetry.d, Raw negative stain EM image of CsgG_(C1S). Arrows indicate thehexadecameric (IV) and octameric (V) particles observed bysize-exclusion chromatography in g. e, Representative class average forside views of CsgG_(C1S) oligomers. No top views were observed for thisconstruct. f, Table of elution volumes (EV) of CsgG_(C1S) and CsgGparticles observed by size-exclusion chromatography shown in g,calculated molecular mass (MWcalc), expected molecular mass (MWCsgG)corresponding CsgG oligomerization state (CsgG_(n)) and the particles'symmetry as observed by negative-stain EM and X-ray crystallography. g,Size-exclusion chromatogram of CsgG_(C1S) (black) andC8E4/LDAO-solubilized CsgG (grey) run on Superdex 200 10/300 GL (GEHealthcare). h, i, Ribbon representation of crystallized oligomers intop and side view, showing the D8 hexadecamers for CsgG_(C1S) (h) and D9octadecamers for membrane-extracted CsgG (i). One protomer is colouredin rainbow from N terminus (blue) to C terminus (red). The two C8octamers (CsgG_(C1S)) or C9 nonamers (CsgG) that form the tail-to-taildimers captured in the crystals are coloured blue and tan. r and h giveradius and interprotomer rotation, respectively.

FIG. 48 shows a comparison of CsgG with structural homologues andinterprotomer contacts in CsgG. a, b, Ribbon diagram for the CsgG_(C1S)monomer (for example CsgG in pre-pore conformation) (a) and thenucleotide-binding-domain-like domain of TolB (b) (PDB 2hqs), bothcoloured in rainbow from N terminus (blue) to C terminus (red). Commonsecondary structure elements are labelled equivalently. c, CsgG_(C1S)(grey) in superimposition with, from left to right, Xanthomonascampestris rare lipoprotein B (PDB 2r76, coloured pink), Shewanellaoneidensis hypothetical lipoprotein DUF330 (PDB 2iqi, coloured pink) andEscherichia coli TolB (PDB 2hqs, coloured pink and yellow for theN-terminal and b-propeller domains, respectively). CsgG-specificstructural elements are labelled and coloured as in the upper leftpanel. d, e, Ribbon diagram of two adjacent protomers as found in theCsgG structure, viewed along the plane of the bilayer, either fromoutside (c) or inside (d) the oligomer. One protomer is shown in rainbow(dark blue to red) from N terminus to C terminus; a second protomer isshown in light blue (core domain), blue (helix 2) and tan (TM domain).Four main oligomerization interfaces are apparent: b6-b39 main-chaininteractions inside the b-barrel, the constriction loop (CL), side-chainpacking of helix 1 (α1) against b1-b3-b4-b5, and helix-helix packing ofhelix 2 (α2). The 18-residue N-terminal loop connecting the lipid anchor(a magenta sphere shows the Ca position of Leu 2) and N-terminal helix(αN) is also seen to wrap over the adjacent two protomers. The projectedposition of the lipid anchor is expected to lie against the TM1 and TM2hairpins of the +2 protomer (not shown for clarity).

FIG. 49 shows Cys accessibility assays for selected surface residues inthe CsgG oligomers. a-c, Ribbon representation of CsgG nonamers shown inperiplasmic (a), side (b) and extracellular (c) views. One protomer iscoloured in rainbow from N terminus (blue) to C terminus (red). Cysteinesubstitutions are labelled and the equivalent locations of the S atomsare shown as spheres, coloured according to accessibility to MAL-PEG(5,000 Da) labelling in E. coli outer membranes. d, Western blot ofMAL-PEG reacted samples analysed on SDS-PAGE, showing 5 kDa increase onMALPEG binding of the introduced cysteine. Accessible (11 and 111),moderately accessible (1) and inaccessible (2) sites are coloured green,orange and red, respectively, in a-e. For Arg 97 and Arg 110 a secondspecies at 44 kDa is present, corresponding to a fraction of protein inwhich both the introduced and native cysteine became labelled. Data arerepresentative of four independent experiments from biologicalreplicates. e, Side view of the dimerization interface in the D9octadecamer as present in the X-ray structure. Introduced cysteines inthe dimerization interface or inside the lumen of the D9 particle arelabelled. In membrane-bound CsgG, these residues are accessible toMAL-PEG, demonstrating that the D9 particles are an artifact ofconcentrated solutions of membrane-extracted CsgG and that the C9complex forms the physiologically relevant species. Residues in theC-terminal helix (aC; Lys 242, Asp 248 and His 255) are found to beinaccessible to poorly accessible, indicating that aC may formadditional contacts with the E. coli cell envelope, possibly thepeptidoglycan layer.

FIG. 50 shows molecular dynamics simulation of CsgG constriction withmodel polyalanine chain, a, b, Top (a) and side (b) views of the CsgGconstriction modeled with a polyalanine chain threaded through thechannel in an extended conformation, here shown in a C-terminal toN-terminal direction. Substrate passage through the CsgG transporter isitself not sequence specific^(ref16,23). For clarity, a polyalaninechain was used for modeling the putative interactions of a passingpolypeptide chain. The modelled area is composed of nine concentric CsgGC-loops, each comprising residues 47-58. Side chains lining theconstriction are shown in stick representation, with Phe 51 colouredslate blue, Asn 55 (amide-clamp) cyan, and Phe 48 and Phe 56 (ϕ-clamp)in light and dark orange, respectively. N, O and H atoms (only hydroxylor side-chain amide H atoms are shown) are coloured blue, red and white,respectively. The polyalanine chain is coloured green, blue, red andwhite for C, N, O and H atoms, respectively. Solvent molecules (water)within 10 Å of the polyalanine residues inside the constriction(residues labelled 11 to 15) are shown as red dots. c, Modelledsolvation of the polyalanine chain, position as in b and with C-loopsremoved for clarity (shown solvent molecules are those within 10 Å ofthe full polyalanine chain). At the height of the amide-clamp andϕ-clamp, the solvation of the polyalanine chain is reduced to a singlewater shell that bridges the peptide backbone and amide-clamp sidechains. Most side chains in the Tyr 51 ring have rotated towards thesolvent in comparison with their inward, centre-pointing positionobserved in the CsgG (and the CsgG_(C1S)) X-ray structure. The model isthe result of a 40 ns all-atom explicit solvent molecular dynamicssimulation with GROMACS^(ref53) using the AMBER99SB-ILDN54 force fieldand with the Cα atoms of the residues at the extremity of the C-loop(Gln 47 and Thr 58) positionally restricted.

FIG. 51 shows sequence conservation in CsgG homologues. a, Surfacerepresentation of the CsgG nonamer coloured according to sequencesimilarity (coloured yellow to blue from low to high conservation score)and viewed from the periplasm (far left), the side (middle left), theextracellular milieu (middle right) or as a cross-sectional side view(far right). The figures show that the regions of highest sequenceconservation map to the entry of the periplasmic vestibule, thevestibular side of the constriction loop and the luminal surface of theTM domain, b, Multiple sequence alignment of CsgGlike lipoproteins. Theselected sequences were chosen from monophyletic clades across thephylogenetic three of CsgG-like sequences (not shown), to give arepresentative view of sequence diversity. Secondary structure elementsare shown as arrows or bars for β-strands and α-helices, respectively,and are based on the E. coli CsgG crystal structure. c, d, CsgG protomerin secondary structure representation (c) and a cross-sectional sideview (d) of the CsgG nonamer in surface representation, both colouredgrey and with three continuous blocks of high sequence conservationcoloured red (HCR1), blue (HCR2) and yellow (HCR3). HCR1 and HCR2 shapethe vestibular side of the constriction loop; HCR3 corresponds to helix2, lying at the entry of the periplasmic vestibule. Inside theconstriction, Phe 56 is 100% conserved, whereas Asn 55 can beconservatively replaced by Ser or Thr, for example by a small polar sidechain that can act as hydrogen-bond donor/acceptor. The concentricside-chain ring at the exit of the constriction (Tyr 51) is notconserved. The presence of the Phe-ring at the entrance of theconstriction is topologically similar to the Phe 427-ring (referred toas the ϕ-clamp) in the anthrax protective antigen PA63, in which it wasshown to catalyse polypeptide capture and passage^(ref20). MST of toxBsuperfamily proteins reveals a conserved motif D(D/Q)(F)(S/N)S at theheight of the Phe-ring. This is similar to the S(Q/N/T)(F)ST motif seenin curti-like transporters. Although an atomic resolution structure ofPA63 in pore conformation is not yet available, available structuressuggest the Phe-ring may similarly be followed by a conservedhydrogen-bond donor/acceptor (Ser/Asn 428) as a subsequent concentricring in the translocation channel (note that the orientation of theelement is inverted in both transporters).

FIG. 52 shows single-channel current analysis of CsgG and CsgG:CsgEpores. a, Under negative field potential, CsgG pores show twoconductance states. The upper left and right panels show arepresentative single-channel current trace of, respectively, the normal(measured at +50, 0 and −50 mV) and the low-conductance forms (measuredat 0, +50 and −50 mV). No conversions between both states were observedduring the total observation time (n=22), indicating that theconductance states have long lifetimes (second to minute timescale). Thelower left panel shows a current histogram for the normal andlow-conductance forms of CsgG pores acquired at +50 and −50 mV (n=33).I-V curves for CsgG pores with regular and low conductance are shown inthe lower right panel. The data represent averages and standarddeviations from at least four independent recordings. The nature orphysiological existence of the low-conductance form is unknown. b,Electrophysiology of CsgG channels titrated with the accessory factorCsgE. The plots display the fraction of open, intermediate and closedchannels as a function of CsgE concentration. Open and closed states ofCsgG are illustrated in FIG. 45f . Increasing the concentration of CsgEto more than 10 nM leads to the closure of CsgG pores. The effect occursat +50 mV (left) and −50 mV (right), ruling out the possibility that thepore blockade is caused by electrophoresis of CsgE (calculated pl 4.7)into the CsgG pore. An infrequent (5%) intermediate state has roughlyhalf the conductance of the open channel. It may represent CsgE-inducedincomplete closures of the CsgG channel; alternatively, it couldrepresent the temporary formation of a CsgG dimer caused by the bindingof residual CsgG monomer from the electrolyte solution to themembrane-embedded pore. The fraction for the three states was obtainedfrom all-point histogram analysis of single-channel current traces. Thehistograms yielded peak areas for up to three states, and the fractionfor a given state was obtained by dividing the corresponding peak areaby the sum of all other states in the recording. Under negative fieldpotential, two open conductance states are discerned, similar to theobservations for CsgG (see a). Because both open channel variations wereblocked by higher CsgE concentrations, the ‘open’ traces in b combineboth conductance forms. The data in the plot represent averages andstandard deviations from three independent recordings. c, The crystalstructure, size-exclusion chromatography and EM show that detergentextracted CsgG pores form non-native tall-to-tall stacked dimers (forexample, two nonamers as D9 particle; FIG. 47) at higher proteinconcentration. These dimers can also be observed in single-channelrecordings. The upper panel shows the single channel current trace of astacked CsgG pore at +50, 0 and −50 mV (left to right). The lower leftpanel shows a current histogram of dimeric CsgG pores recorded at +50and −50 mV. The experimental conductances of +16.2±1.8 and −16.0±3.0 pA(n=15) at +50 and −50 mV, respectively, are near the theoreticallycalculated value of 23 pA. The lower right panel shows an I-V curve forthe stacked CsgG pores. The data represent averages and standarddeviations from six independent recordings. d, The ability of CsgE tobind and block stacked CsgG pores was tested by electrophysiology. Shownare single-channel current traces of stacked CsgG pore in the presenceof 10 or 100 nM CsgE at +50 mV (upper) and −50 mV (lower). The currenttraces indicate that otherwise saturating concentrations of CsgE do notlead to pore closure for stacked CsgG dimers. These observations are ingood agreement with the mapping of the CsgG-CsgE contact zone to helix 2and the mouth of the CsgG periplasmic cavity as discerned by EM andsite-directed mutagenesis (FIG. 45 and FIG. 52).

FIG. 53 shows CsgE oligomer and CsgG-CsgE complex. a, Size exclusionchromatography of CsgE (Superose 6, 16/600; running buffer 20 mMTris-HCl pH8, 100 mM NaCl, 2.5% glycerol) shows an equilibrium of twooligomeric states, 1 and 2, with an apparent molecular mass ratio of9.16:1. Negative-stain EM inspection of peak 1 shows discrete CsgEparticles (five representative class averages are shown in the inset,ordered by increasing tilt angles) compatible in size with nine CsgEcopies. b, Selected class average of CsgE oligomer observed in top viewby cryo-EM and its rotational autocorrelation show the presence of C9symmetry. c, FSC analysis of CsgG-CsgE cryo-EMmodel. Three-dimensionalreconstruction achieved a resolution of 24 Å as determined by FSC at athreshold of 0.5 correlation using 125 classes corresponding to 1,221particles. d, Overlay of CsgG-CsgE cryo-EM density and the CsgG nonamerobserved in the X-ray structure. The overlays are shown viewed from theside as semi-transparent density (left) or as across-sectional view. e,Congo red binding of E. coli BW25141ΔcsgG complemented with wild-typecsgG (WT), empty vector (ΔcsgG) or csgGhelix 2 mutants (single aminoacid replacements labelled in single-letter code). Data arerepresentative of four biological replicates. f, Effect of bile salttoxicity on E. coli LSR12 complemented with csgG (WT) or on csgGcarrying different helix 2 mutations, complemented with (1) or without(2) csgE. Tenfold serial dilution starting from 107 bacteria werespotted on McConkey agar plates. Expression of the CsgG pore in theouter membrane leads to an increased bile salt sensitivity that can beblocked by co-expression of CsgE (n=6, three biological replicates, withtwo repetitions each). g, Cross-sectional view of CsgG X-ray structurein molecular surface representation. CsgG mutants without an effect onCongo red binding or toxicity are shown in blue; mutants that interferewith CsgE-mediated rescue of bile salt sensitivity are indicated in red.

FIG. 54 shows assembly and substrate recruitment of the CsgG secretioncomplex. The curli transporter CsgG and the soluble secretion cofactorCsgE form a secretion complex with 9:9 stoichiometry that encloses a,24,000 Å³ chamber that is proposed to entrap the CsgA substrate andfacilitate its entropy-driven diffusion across the outer membrane (OM;see the text and FIG. 45). On theoretical grounds, three putativepathways (a-c) for substrate recruitment and assembly of the secretioncomplex can be envisaged. a, A ‘catch-and-cap’ mechanism entails thebinding of CsgA to the apo CsgG translocation channel (1), leading to aconformational change in the latter that exposes a high-affinity bindingplatform for CsgE binding (2). CsgE binding leads to capping of thesubstrate cage. On secretion of CsgA, CsgG would fall back into itslow-affinity conformation, leading to CsgE dissociation and liberationof the secretion channel for a new secretion cycle. b, In a ‘dockand-trap’ mechanism, periplasmic CsgA is first captured by CsgE (1),causing the latter to adopt a high-affinity complex that docks onto theCsgG translocation pore (2), enclosing CsgA in the secretion complex.CsgA binding could be directly to CsgE oligomers or to CsgE monomers,the latter leading to subsequent oligomerization and CsgGbinding.Secretion of CsgA leads CsgE to fall back into its low-affinityconformation and to dissociate from the secretion channel. c, CsgG andCsgE form a constitutive complex, in which CsgE conformational dynamicscycle between open and closed forms in the course of CsgA recruitmentand secretion. Currently published or available data do not allow us todiscriminate between these the putative recruitment modes or derivativesthereof, or to put forward one of them.

FIG. 55 shows data collection statistics and electron density maps ofCsgG_(C1S) and CsgG. a, Data collection statistics for CsgG_(C1S) andCsgG X-ray structures. b, Electron density map at 2.8 Å for CsgG_(C1S)calculated using NCS-averaged and density-modified experimental SADphases, and contoured at 1.5σ. The map shows the region of the channelconstruction (CL; a single protomer is labeled) and is overlaid on thefinal refined model. c, Electron density map (resolutions 3.6, 3.7 and3.8 Å along reciprocal vectors a*, b* and c*, respectively) in the CsgGTM domain region, calculated from NCS averaged and density-modifiedmolecular replacement phases (TM loops were absent from the inputmodel); B-factor sharpened by −20 Å² and contoured at 1.0σ. The figureshows the TM1 (Lys 135-Leu 154) and TM2 (Leu 182-Asn 209) region of asingle CsgG protomer, overlaid on the final refined model.

FIG. 56 shows a single channel current trace (left) and zoomed regionthereof (right) of a CsgG WT protein interacting with a DNA hairpincarrying a single-stranded DNA overhang. The trace shows the currentwhich alters in response to the potential measured at +50 mV or −50 mVintervals (indicated by arrows). The downward current blockades in thelast +50 mV segment represent the simultaneous lodging of the hairpinduplex inside the pore lumen and threading of the single-strandedhairpin end into inner pore construction leading to an almost completecurrent blockade. Reversal of the electrical field to −50 mV results inthe electrophoretic unblocking of the pore. A new +50 mV episode resultsagain in DNA hairpin lodging/threading and pore blockage. On the +50 mVsegments, unfolding of the hairpin structure can lead to the terminationof the current blockade indicated by the reversal of the currentblockade. The hairpin with the sequence was 3′ GCGGGGA GCGTATT AGAGTTGGATCGGATGCA GCTGGCTACTGACGTCA TGACGTCAGTAGCCAGCATGCATCCGATC-5′ was addedto the cis side of the chamber at a final concentration of 10 nM.

FIG. 57 shows the purification and channel properties of CsgG-ΔPYPA, amutant CsgG pore where the PYPA sequence (residues 50-53) at theconstriction residue Y51 is mutated to GG.

DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 shows amino acid sequence of wild-type E. coli CsgGincluding signal sequence (Uniprot accession number P0AEA2).

SEQ ID NO: 2 shows polynucleotide sequence of wild-type E. coli CsgGincluding signal sequence (Gene ID: 12932538).

SEQ ID NO: 3 shows the amino acid sequence of the wild-type E. coli CsgGfrom positions 53 to 77 of SEQ ID NO:2. This corresponds to the aminoacid sequence from positions 38 to 63 of the mature wild-type E. coliCsgG monomer (i.e. lacking the signal sequence).

SEQ ID NOs 4 to 388 show the amino acid sequence from positions 38 to 63of the modified monomers of CsgG lacking the signal sequence.

SEQ ID NO: 389 shows the codon optimised polynucleotide sequenceencoding the wild-type CsgG monomer from Escherichia coli Str. K-12substr. MC4100. This monomer lacks the signal sequence.

SEQ ID NO: 390 shows the amino acid sequence of the mature form of thewild-type CsgG monomer from Escherichia coli Str. K-12 substr. MC4100.This monomer lacks the signal sequence. The abbreviation used for thisCsgG=CsgG-Eco.

SEQ ID NO: 391 shows the amino acid sequence of YP_001453594.1: 1-248 ofhypothetical protein CKO_02032 [Citrobacter koseri ATCC BAA-895], whichis 99% identical to SEQ ID NO: 390.

SEQ ID NO: 392 shows the amino acid sequence of WP_001787128.1: 16-238of curli production assembly/transport component CsgG, partial[Salmonella enterica], which is 98% to SEQ ID NO: 390.

SEQ ID NO: 393 shows the amino acid sequence of KEY44978.1|: 16-277 ofcurli production assembly/transport protein CsgG [Citrobacteramalonaticus], which is 98% identical to SEQ ID NO: 390.

SEQ ID NO: 394 shows the amino acid sequence of YP_003364699.1: 16-277of curli production assembly/transport component [Citrobacter rodentiumICC168], which is 97% identical to SEQ ID NO: 390.

SEQ ID NO: 395 shows the amino acid sequence of YP_004828099.1: 16-277of curli production assembly/transport component CsgG [Enterobacterasburiae LF7a], which is 94% identical to SEQ ID NO: 390.

SEQ ID NO: 396 shows the polynucleotide sequence encoding the Phi29 DNApolymerase.

SEQ ID NO: 397 shows the amino acid sequence of the Phi29 DNApolymerase.

SEQ ID NO: 398 shows the codon optimised polynucleotide sequence derivedfrom the sbcB gene from E. coli. It encodes the exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 399 shows the amino acid sequence of exonuclease I enzyme(EcoExo I) from E. coli.

SEQ ID NO: 400 shows the codon optimised polynucleotide sequence derivedfrom the xthA gene from E. coli. It encodes the exonuclease III enzymefrom E. coli.

SEQ ID NO: 401 shows the amino acid sequence of the exonuclease IIIenzyme from E. coli. This enzyme performs distributive digestion of 5′monophosphate nucleosides from one strand of double stranded DNA (dsDNA)in a 3′-5′ direction. Enzyme initiation on a strand requires a 5′overhang of approximately 4 nucleotides.

SEQ ID NO: 402 shows the codon optimised polynucleotide sequence derivedfrom the recJ gene from T. thermophilus. It encodes the RecJ enzyme fromT. thermophilus (TthRecJ-cd).

SEQ ID NO: 403 shows the amino acid sequence of the RecJ enzyme from T.thermophilus (TthRecJ-cd). This enzyme performs processive digestion of5′ monophosphate nucleosides from ssDNA in a 5′-3′ direction. Enzymeinitiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 404 shows the codon optimised polynucleotide sequence derivedfrom the bacteriophage lambda exo (redX) gene. It encodes thebacteriophage lambda exonuclease.

SEQ ID NO: 405 shows the amino acid sequence of the bacteriophage lambdaexonuclease. The sequence is one of three identical subunits thatassemble into a trimer. The enzyme performs highly processive digestionof nucleotides from one strand of dsDNA, in a 5′-3′direction(http://www.neb.com/nebecomm/products/productM0262.asp). Enzymeinitiation on a strand preferentially requires a 5′ overhang ofapproximately 4 nucleotides with a 5′ phosphate.

SEQ ID NO: 406 shows the amino acid sequence of Hel308 Mbu.

SEQ ID NO: 407 shows the amino acid sequence of Hel308 Csy.

SEQ ID NO: 408 shows the amino acid sequence of Hel308 Tga.

SEQ ID NO: 409 shows the amino acid sequence of Hel308 Mhu.

SEQ ID NO: 410 shows the amino acid sequence of Tral Eco.

SEQ ID NO: 411 shows the amino acid sequence of XPD Mbu.

SEQ ID NO: 412 shows the amino acid sequence of Dda 1993.

SEQ ID NO: 413 shows the amino acid sequence of Trwc Cba.

SEQ ID NO: 414 shows the amino acid sequence of WP_006819418.1: 19-280of transporter [Yokenella regensburgei], which is 91% identical to SEQID NO: 390.

SEQ ID NO: 415 shows the amino acid sequence of WP_024556654.1: 16-277of curli production assembly/transport protein CsgG [Cronobacterpulveris], which is 89% identical to SEQ ID NO: 390.

SEQ ID NO: 416 shows the amino acid sequence of YP_005400916.1:16-277 ofcurli production assembly/transport protein CsgG [Rahnella aquatilisHX2], which is 84% identical to SEQ ID NO: 390.

SEQ ID NO: 417 shows the amino acid sequence of KFC99297.1: 20-278 ofCsgG family curli production assembly/transport component [Kluyveraascorbata ATCC 33433], which is 82% identical to SEQ ID NO: 390.

SEQ ID NO: 418 shows the amino acid sequence of KFC86716.1|:16-274 ofCsgG family curli production assembly/transport component [Hafnia alveiATCC 13337], which is 81% identical to SEQ ID NO: 390.

SEQ ID NO: 419 shows the amino acid sequence of YP_007340845.1|:16-270of uncharacterised protein involved in formation of curli polymers[Enterobacteriaceae bacterium strain FGI 57], which is 76% identical toSEQ ID NO: 390.

SEQ ID NO: 420 shows the amino acid sequence of WP_010861740.1: 17-274of curli production assembly/transport protein CsgG [Plesiomonasshigelloides], which is 70% identical to SEQ ID NO: 390.

SEQ ID NO: 421 shows the amino acid sequence of YP_205788.1: 23-270 ofcurli production assembly/transport outer membrane lipoprotein componentCsgG [Vibrio fischeri ES114], which is 60% identical to SEQ ID NO: 390.

SEQ ID NO: 422 shows the amino acid sequence of WP_017023479.1: 23-270of curli production assembly protein CsgG [Aliivibrio logei], which is59% identical to SEQ ID NO: 390.

SEQ ID NO: 423 shows the amino acid sequence of WP_007470398.1: 22-275of Curli production assembly/transport component CsgG [Photobacteriumsp. AK15], which is 57% identical to SEQ ID NO: 390.

SEQ ID NO: 424 shows the amino acid sequence of WP_021231638.1: 17-277of curli production assembly protein CsgG [Aeromonas veronii], which is56% identical to SEQ ID NO: 390.

SEQ ID NO: 425 shows the amino acid sequence of WP_033538267.1: 27-265of curli production assembly/transport protein CsgG [Shewanella sp.ECSMB14101], which is 56% identical to SEQ ID NO: 390.

SEQ ID NO: 426 shows the amino acid sequence of WP_003247972.1: 30-262of curli production assembly protein CsgG [Pseudomonas putida], which is54% identical to SEQ ID NO: 390.

SEQ ID NO: 427 shows the amino acid sequence of YP_003557438.1: 1-234 ofcurli production assembly/transport component CsgG [Shewanella violaceaDSS12], which is 53% identical to SEQ ID NO: 390.

SEQ ID NO: 428 shows the amino acid sequence of WP_027859066.1: 36-280of curli production assembly/transport protein CsgG [Marinobacteriumjannaschii], which is 53% identical to SEQ ID NO: 390.

SEQ ID NO: 429 shows the amino acid sequence of CEJ70222.1: 29-262 ofCurli production assembly/transport component CsgG [Chryseobacteriumoranimense G311], which is 50% identical to SEQ ID NO: 390.

SEQ ID NO: 430 shows a polynucleotide sequence used in Example 18.

SEQ ID NO: 431 shows a polynucleotide sequence used in Example 18.

SEQ ID NO: 432 shows a polynucleotide sequence used in Example 18.

SEQ ID NO: 433 shows a polynucleotide sequence used in Example 18.

SEQ ID NO: 434 shows a polynucleotide sequence used in Example 18.Attached to the 3′ end of SEQ ID NO: 434 is six iSp18 spacers which areattached at the opposite end to two thymines and a 3′ cholesterol TEG.

SEQ ID NO: 435 shows the amino acid sequence of StepII(C).

SEQ ID NO: 436 shows the amino acid sequence of Pro.

SEQ ID NO: 437 to 440 show primers from example 1.

SEQ ID NO: 441 shows the Hairpin from example 21.

SEQ ID NO: 442 to 448 show the sequences from FIG. 4.

SEQ ID NO: 449 and 450 show the sequences from FIG. 43.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, the practice of the present inventionemploys conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA technology, and chemical methods, whichare within the capabilities of a person of ordinary skill in the art.Such techniques are also explained in the literature, for example, M. R.Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, FourthEdition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel, F. M. et al. (1995 and periodic supplements;Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley &Sons, New York, N.Y.); B. Roe, J. Crabtree, and A. Kahn, 1996, DNAIsolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M.Polak and James O'D. McGee, 1990, In Situ Hybridisation: Principles andPractice, Oxford University Press; M. J. Gait (Editor), 1984,Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D. M. J.Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA StructurePart A: Synthesis and Physical Analysis of DNA Methods in Enzymology,Academic Press. Each of these general texts is herein incorporated byreference.

Prior to setting forth the invention, a number of definitions areprovided that will assist in the understanding of the invention. Allreferences cited herein are incorporated by reference in their entirety.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

As used herein, the term “comprising” means any of the recited elementsare necessarily included and other elements may optionally be includedas well. “Consisting essentially of” means any recited elements arenecessarily included, elements that would materially affect the basicand novel characteristics of the listed elements are excluded, and otherelements may optionally be included. “Consisting of” means that allelements other than those listed are excluded. Embodiments defined byeach of these terms are within the scope of this invention.

The term “nucleic acid” as used herein, is a single or double strandedcovalently-linked sequence of nucleotides in which the 3′ and 5′ ends oneach nucleotide are joined by phosphodiester bonds. The polynucleotidemay be made up of deoxyribonucleotide bases or ribonucleotide bases.Nucleic acids may include DNA and RNA, and may be manufacturedsynthetically in vitro or isolated from natural sources. Nucleic acidsmay further include modified DNA or RNA, for example DNA or RNA that hasbeen methylated, or RNA that has been subject to post-translationalmodification, for example 5′-capping with 7-methylguanosine,3′-processing such as cleavage and polyadenylation, and splicing.Nucleic acids may also include synthetic nucleic acids (XNA), such ashexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threosenucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid(LNA) and peptide nucleic acid (PNA). Sizes of nucleic acids, alsoreferred to herein as “polynucleotides” are typically expressed as thenumber of base pairs (bp) for double stranded polynucleotides, or in thecase of single stranded polynucleotides as the number of nucleotides(nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides ofless than around 40 nucleotides in length are typically called“oligonucleotides” and may comprise primers for use in manipulation ofDNA such as via polymerase chain reaction (PCR).

The term “amino acid” in the context of the present invention is used inits broadest sense and is meant to include naturally occurring L α-aminoacids or residues. The commonly used one and three letter abbreviationsfor naturally occurring amino acids are used herein: A=Ala; C=Cys;D=Asp; E=Glu; F=Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M=Met; N=Asn;P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger,A. L., (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, NewYork). The general term “amino acid” further includes D-amino acids,retro-inverso amino acids as well as chemically modified amino acidssuch as amino acid analogues, naturally occurring amino acids that arenot usually incorporated into proteins such as norleucine, andchemically synthesised compounds having properties known in the art tobe characteristic of an amino acid, such as β-amino acids. For example,analogues or mimetics of phenylalanine or proline, which allow the sameconformational restriction of the peptide compounds as do natural Phe orPro, are included within the definition of amino acid. Such analoguesand mimetics are referred to herein as “functional equivalents” of therespective amino acid. Other examples of amino acids are listed byRoberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Grossand Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., N.Y. 1983,which is incorporated herein by reference.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or in vitro by synthetic means.Polypeptides of less than around 12 amino acid residues in length aretypically referred to as “peptides” and those between about 12 and about30 amino acid residues in length may be referred to as “oligopeptides”.The term “polypeptide” as used herein denotes the product of a naturallyoccurring polypeptide, precursor form or proprotein. Polypeptides canalso undergo maturation or post-translational modification processesthat may include, but are not limited to: glycosylation, proteolyticcleavage, lipidization, signal peptide cleavage, propeptide cleavage,phosphorylation, and such like. The term “protein” is used herein torefer to a macromolecule comprising one or more polypeptide chains.

A “biological pore” is a trans-membrane protein structure defining achannel or hole that allows the translocation of molecules and ions fromone side of the membrane to the other. The translocation of ionicspecies through the pore may be driven by an electrical potentialdifference applied to either side of the pore. A “nanopore” is abiological pore in which the minimum diameter of the channel throughwhich molecules or ions pass is in the order of nanometres (10⁻⁹metres).

For all aspects and embodiments of the present invention, apolynucleotide can comprise a polynucleotide that has at least 50%, 60%,70%, 80%, 90%, 95% or 99% complete sequence identity to wild-type E.coli CsgG as shown in SEQ ID NO: 2. Likewise, the polypeptide cancomprise a polypeptide that has at least 50%, 60%, 70%, 80%, 90%, 95% or99% complete sequence identity to wild-type E. coli CsgG as shown in SEQID NO: 1. A polypeptide can comprise a polypeptide that contains thePFAM domain PF03783, which is characteristic for CsgG-like proteins. Alist of presently known CsgG homologues and CsgG architectures can befound at http://pfam.xfam.org//family/PF03783. Sequence identity canthus also be to a fragment or portion of the full length polynucleotideor polypeptide. Hence, a sequence may have only 50% overall sequenceidentity with a sequence of the invention but a particular region,domain or subunit could share 80%, 90%, or as much as 99% sequenceidentity with sequences of the invention. According to the presentinvention, homology to the nucleic acid sequence of SEQ ID NO: 2 is notlimited simply to sequence identity. Many nucleic acid sequences candemonstrate biologically significant homology to each other despitehaving apparently low sequence identity. In the present inventionhomologous nucleic acid sequences are considered to be those that willhybridise to each other under conditions of low stringency (M. R. Green,J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, FourthEdition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

The term “vector” is used to denote a DNA molecule that is either linearor circular, into which another nucleic acid (typically DNA) sequencefragment of appropriate size can be integrated. Such DNA fragment(s) caninclude additional segments that provide for transcription of a geneencoded by the DNA sequence fragment. The additional segments caninclude and are not limited to: promoters, transcription terminators,enhancers, internal ribosome entry sites, untranslated regions,polyadenylation signals, selectable markers, origins of replication andsuch like. A variety of suitable promoters for prokaryotic (e.g., the[beta]-lactamase and lactose promoter systems, alkaline phosphatase, thetryptophan (trp) promoter system, lac, tac, T3, T17 promoters for E.coli) and eukaryotic (e.g., simian virus 40 early or late promoter, Roussarcoma virus long terminal repeat promoter, cytomegalovirus promoter,adenovirus late promoter, EG-1a promoter) hosts are available.Expression vectors are often derived from plasmids, cosmids, viralvectors and yeast artificial chromosomes; vectors are often recombinantmolecules containing DNA sequences from several sources. Specificembodiments of the present invention provide for an expression vectorthat encodes a wild type or modified CsgG polypeptide as describedherein. The term “operably linked”, when applied to DNA sequences, forexample in an nucleic acid vector such as mentioned above, indicatesthat the sequences are arranged so that they function cooperatively inorder to achieve their intended purposes, i.e. a promoter sequenceallows for initiation of transcription that proceeds through anassociated coding sequence as far as a termination sequence.

The trans-membrane protein structure of a biological pore may bemonomeric or oligomeric in nature. Typically, the pore comprises aplurality of polypeptide subunits arranged around a central axis therebyforming a protein-lined channel that extends substantially perpendicularto the membrane in which the nanopore resides. The number of polypeptidesubunits is not limited. Typically, the number of subunits is from 5 toup to 30, suitably the number of subunits is from 6 to 10.Alternatively, the number of subunits is not defined as in the case ofperfringolysin or related large membrane pores. The portions of theprotein subunits within the nanopore that form protein-lined channeltypically comprise secondary structural motifs that may include one ormore trans-membrane β-barrel, and/or α-helix sections.

It is to be understood that different applications of the disclosedproducts and methods may be tailored to the specific needs in the art.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments of the invention only, andis not intended to be limiting.

In addition as used in this specification and the appended claims, thesingular forms “a”, “an”, and “the” Include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “apolynucleotide” includes two or more polynucleotides, reference to “apolynucleotide binding protein” includes two or more such proteins,reference to “a helicase” includes two or more helicases, reference to“a monomer” refers to two or more monomers, reference to “a pore”includes two or more pores and the like.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

The present invention relates in part to the bacterial amyloid secretionchannel (CsgG), its method of manufacture and its use in nucleic acidsequencing applications, and molecular sensing.

CsgG is a membrane lipoprotein present in the outer membrane of E. coli(Uniprot accession no. P0AEA2; Gene ID: 12932538). In the outer lipidmembrane, CsgG forms a nanopore comprising an oligomeric complex of nineCsgG monomer subunits. By virtue of the type II (lipoprotein) signalsequence, the CsgG preprotein is translocated across the SEC transloconand subsequently becomes triacylated at the N-terminal Cys residue ofthe mature CsgG (i.e. CsgG with cleaved type II signal sequence).Triacylated, or “lipidated” CsgG is transported to the outer membrane ofa Gram-negative host, where it inserts into the bilayer as a nonamericpore. A non-lipidated form of CsgG, e.g. CsgG_(C1S), exists in theperiplasm as a soluble protein in a pre-pore conformation (FIG. 42).

The X-ray structure of the wild type CsgG nanopore (Goyal et al.,Nature, 2014, 516(7530), 250-3) shows that it has a width of 120 Å and aheight of 85 Å (hereinafter, the term “width” of the nanopore willrelate to its dimension parallel with the membrane surface, and the term“height” of the nanopore will relate to its dimension perpendicular tothe membrane). The CsgG pore complex traverses the membrane through a36-stranded β-barrel to provide a 40 Å inner diameter channel (FIG. 1).The assembled monomers of the CsgG channel each possess a conserved12-residue loop (C-loop, “CL”; FIG. 2), which co-operate so as to form aconstriction in the channel to a diameter of approximately 9.0 Å (FIGS.1 and 2). The constriction in the wild type CsgG nanopore is composed ofthree stacked concentric rings formed by the side chains of amino acidresidues Tyr51, Asn55 and Phe56 of each of the CsgG monomers present inthe CsgG oligomer (FIG. 3). This numbering of residues is based upon themature protein which lacks the native 15 amino acid signal sequence atthe N-terminal. The mature protein therefore corresponds to residues 16to 277 of SEQ ID NO:1. Tyr51 is at position 66 in SEQ ID NO:1, Asn is atposition 70 in SEQ ID NO:1 and Phe56 is at position 71 in SEQ ID NO:1.

The constriction acts to limit the passage of ions and other moleculesthrough the CsgG channel. Single-channel current recordings of CsgGreconstituted in planar phospholipid bilayers led to a steady current of43.1±4.5 pA (n=33) or −45.1±4.0 pA (n=13) using standard electrolyteconditions and a potential of +50 mV or −50 mV, respectively (FIG. 5).

Current flow through the CsgG channel can be effectively blocked by theaddition of stoichiometric quantities of the periplasmic factor CsgE(Uniprot accession no. POAE95; Examples 10 to 12). Without wishing to bebound by theory, current evidence points to a mechanism whereby CsgEforms a complex with the CsgG pore acting to cap one end of the channel.The significant reduction in the flow of ions through the CsgG channelmay be measured using standard single-channel recording techniques(Examples 12 and 13, FIG. 6). The inventors have found that measuredparameters for the current flow (maximum current, and ability to monitorcurrent variation) render the nanopore suitable for use in nucleic acidsequencing and molecular sensing applications according to oneembodiment of the invention.

Accordingly, the present invention relates in part to methods and usesof the CsgG nanopore protein complex in nucleic acid sequencing based onvariations of electrical measurements of the current flowing through ananopore.

Nucleic acids are particularly suitable for nanopore sequencing. Thenaturally-occurring nucleic acid bases in DNA and RNA may bedistinguished by their physical size. As a nucleic acid molecule, orindividual base, passes through the channel of a nanopore, the sizedifferential between the bases causes a directly correlated reduction inthe ion flow through the channel. The variation in ion flow may berecorded. Suitable electrical measurement techniques for recording ionflow variations are described in, for example, WO 2000/28312 and D.Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, pp 7702-7 (singlechannel recording equipment); and, for example, in WO 2009/077734(multi-channel recording techniques). Through suitable calibration, thecharacteristic reduction in ion flow can be used to identify theparticular nucleotide and associated base traversing the channel inreal-time.

The size of the narrowest constriction in a transmembrane channel istypically a key factor in determining suitability of a nanopore fornucleic acid sequencing applications. If the constriction is too small,the molecule to be sequenced will not be able to pass through. However,to achieve a maximal effect on ion flow through the channel, at itsnarrowest point (i.e. at a constriction) the channel should not be toolarge. Ideally, any constriction should be as close as possible indiameter to the size of the base passing through. For sequencing ofnucleic acids and nucleic acid bases, suitable constriction diametersare in the nanometre range (10⁻⁹ metre range). Suitably, the diametershould be in the region of 0.5 to 1.5 nm, typically, the diameter is inthe region of 0.7 to 1.2 nm. The constriction in wild type E. coli CsgGhas a diameter of approximately 9 Å (0.9 nm). The inventors have deducedthat the size and configuration of the constriction in the CsgG channelis suitable for nucleic acid sequencing.

For applications related to nucleic acid sequencing, the CsgG nanoporemay be used in wild-type form or may be further modified, such as bydirected mutagenesis of particular amino acid residues, to furtherenhance the desired properties of the nanopore in use. For example, inembodiments of the present invention mutations are contemplated to alterthe number, size, shape, placement or orientation of the constrictionwithin the channel. Modified mutant CsgG nanopore complex may beprepared by known genetic engineering techniques that result in theinsertion, substitution and/or deletion of specific targeted amino acidresidues in the polypeptide sequence. In the case of the oligomeric CsgGnanopore, the mutations may be made in each monomeric polypeptidesubunit, or any one of the monomers, or all of the monomers. Suitably,in one embodiment of the invention the mutations described are made toall monomeric polypeptides within the oligomeric protein structure.

According to an embodiment of the invention, a modified mutant CsgGnanopore is provided where the number of channel constrictions withinthe pore is reduced.

The wild type E. coli CsgG pore includes two channel constrictions (seeFIG. 1). These are formed by (i) amino acid residues Phe56 and Asn55,and (ii) amino acid residue Tyr 51, as part of a wider structurecomprising additional amino acids from position 54 and to 53, as well asthe C-loop motif (FIGS. 2 and 3).

In typical nanopore nucleic acid sequencing, the open-channel ion flowis reduced as the individual nucleotides of the nucleic sequence ofinterest sequentially pass through the channel of the nanopore due tothe partial blockage of the channel by the nucleotide. It is thisreduction in ion flow that is measured using the suitable recordingtechniques described above. The reduction in ion flow may be calibratedto the reduction in measured ion flow for known nucleotides through thechannel resulting in a means for determining which nucleotide is passingthrough the channel, and therefore, when done sequentially, a way ofdetermining the nucleotide sequence of the nucleic acid passing throughthe nanopore. For the accurate determination of individual nucleotides,it has typically required for the reduction in ion flow through thechannel to be directly correlated to the size of the individualnucleotide passing through the single constriction (or “reading head”).It will be appreciated that sequencing may be performed upon an intactnucleic acid polymer that is ‘threaded’ through the pore via the actionof an associated polymerase, for example. Alternatively, sequences maybe determined by passage of nucleotide triphosphate bases that have beensequentially removed from a target nucleic acid in proximity to the pore(see for example WO 2014/187924).

When two or more constrictions are present and spaced apart eachconstriction may interact or “read” separate nucleotides within thenucleic acid strand at the same time. In this situation, the reductionin ion flow through the channel will be the result of the combinedrestriction in flow of all the constrictions containing nucleotides.Hence, in some instances a double constriction may lead to a compositecurrent signal. In certain circumstances, the current read-out for oneconstriction, or “reading head”, may not be able to be determinedindividually when two such reading heads are present.

The wild-type pore structure of CsgG may be re-engineered viarecombinant genetic techniques to widen, alter, or remove one of the twoconstrictions to leave a single constriction within the channel, thus,defining a single reading head. The constriction motif in the CsgGoligomeric pore is located at amino acid residues at position 38 to 63in the wild type monomeric E. coli CsgG polypeptide. The wild-type aminoacid sequence of this region is provided as SEQ ID NO: 3. In consideringthis region, mutations at any of the amino acid residue positions 50 to53, 54 to 56 and 58 to 59 are contemplated as within the remit of thepresent invention. Based on sequence similarity with CsgG homologues(FIG. 4), amino acid residue positions 38 to 49, 53, 57, and 61 to 63are considered to be highly conserved and therefore may be less suitablefor substitution or other modification. Due to the key positioning ofthe sidechains of Tyr51, Asn55, and Phe56 within the channel of thewild-type CsgG structure, mutation at these positions may beadvantageous in order to modify or alter the characteristics of thereading head.

Mutations at a given position of the monomeric CsgG protein may resultin the substitution of the wild-type amino acid at that position withany other natural or unnatural amino acid. In one embodiment of theinvention, it is desirable to widen or remove a constriction; suitablythe amino acid sidechain in the modified CsgG protein will be selectedso as to be less sterically encumbering than the amino acid sidechain inthe wild type structure which it replaces. The replacement amino acidresidue at a given position may have similar electrostatic properties,or it may have different electrostatic properties. Suitably, thereplacement amino acid sidechain will possess a similar electrostaticcharge to the amino acid sidechain in the wild type structure which itreplaces in order to minimise disruption to secondary structure or theproperties of the channel.

The selection of replacement amino acid may be based on a BLOSUM62matrix which provides a standard methodology for calculating thelikelihood of an amino acid being substituted for another based on alarge multiple sequence alignment. Examples of BLOSUM62 matrices arefreely available to the skilled person on the internet; see for examplethe website of the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/Class/Structure/aa/aa_explorer.cgi).

For Tyr51 in the wild-type CsgG structure, which acts to form a firstconstriction in the CsgG channel, substitution with any amino acid isprovided. In particular, in certain embodiments of the invention Tyr51may be substituted with alanine, glycine, valine, leucine, isoleucine,asparagine, glutamine, and phenylalanine (SEQ ID: 39-318). Inembodiments of the invention, substitution of Tyr51 with alanine orglycine is particularly suitable (SEQ ID: 39-108). In embodiments,residues 50 to 53 (PYPA in the wild-type sequence) may be replaced withglycine-glycine (GG) (SEQ ID: 354-388).

For Asn55, which contributes to the second constriction in the CsgGchannel, substitution with any amino acid is provided. In particular, incertain embodiments of the invention Asn55 may be substituted withalanine, glycine, valine, serine or threonine (SEQ ID: 9-33, 44-68,79-103 114-138, 149-173, 184-208, 219-243, 254-278, 289-313 and324-348).

For Phe56 which forms part of the second constriction in the CsgGchannel, substitution with any amino acid is provided. In particular, incertain embodiments of the invention Phe56 may be substituted withalanine, glycine, valine, leucine, isoleucine, asparagine, and glutamine(SEQ ID: 5-13, 15-18, 20-23, 25-28, 30-33, 40-43, 45-48, 50-53, 55-58,60-63, 65-68, 75-78, 80-83, 85-88, 90-93, 95-98, 100-103, 110-113,115-118, 120-123, 125-128, 130-133, 135-138, 145-148, 150-153, 155-158,160-163, 165-168, 170-173, 180-183, 185-188, 190-193, 195-198, 200-203,205-208, 215-218, 220-223, 225-228, 230-233, 235-238, 240-243, 250-253,255-258, 260-263, 265-268, 270-273, 275-578, 285-288, 290-293, 295-298,300-303, 305-308, 310-313, 320-323, 325-328, 330-333, 335-338, 340-343and 345-348). In embodiments of the invention, substitution of Phe56with alanine and glycine is particularly suitable (SEQ ID: 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380,385, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, 66, 71, 76, 81, 86,91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161,166, 171, 176, 181, 186, 191, 196, 201, 206, 211, 216, 221, 226, 231,236, 241, 246, 251, 256, 261, 266, 271, 276, 281, 286, 291, 296, 301,306, 311, 316, 321, 326, 331, 336, 341 346, 351, 356, 361, 366, 371,376, 381 and 386).

In a given mutant CsgG protein, substitution of Tyr51 may be performedat the same time as either one of positions 55 and 56 (SEQ ID: 44, 49,54, 59, 64, 79, 84, 89, 94, 99, 114, 119, 124, 129, 134, 149, 154, 159,164, 169, 184, 189, 194, 199, 204, 219, 224, 229, 234, 239, 254, 259,264, 269, 274, 289, 294, 299, 304, 309, 359, 364, 369, 374, 379, 40, 41,42, 75, 76 77, 110, 111, 112, 145, 146, 147, 180, 181, 182, 215, 216,217, 250, 251, 252, 285, 286 and 287), wherein at least one constrictionof suitable dimensions within the channel is maintained. Alternatively,substitution of Tyr51 is mutually exclusive to substitution at both ofpositions Asn55 and Phe56 (SEQ ID: 39, 74, 109, 144, 179, 214, 249, 284,354, 10, 11, 12, 15, 16, 17, 20, 21, 22, 25, 26, 27, 30, 31 and 32

Alternatively, one or more of the Tyr51, Asn55 or Phe56 in the wild typeCsgG protein may be deleted (SEQ ID: 319-353, 34-38, 69-73, 104-108,139-143, 174-178, 209-213, 244-248, 279-283, 314-318, 384-388, 8, 13,18, 23, 28, 33, 43, 48, 53, 58, 63, 68, 78, 83, 88, 93, 98, 103, 113,118, 123, 128, 133, 138, 148, 153, 158, 163, 168, 173, 183, 188, 193,198, 203, 208, 218, 223, 228, 233, 238, 243, 253, 258, 263, 268, 273,278, 288, 293, 298, 303, 308 and 313). To maintain at least oneconstriction in the channel, in a given embodiment, deletion of aminoacid residue Tyr51 is mutually exclusive to deletion of both amino acidresidues Asn55 and Phe56 (SEQ ID: 319-322, 324-327, 329-332, 334-337,339-342, 344-347, 38, 73, 108, 143, 178, 213, 248, 283 and 318). Certainneighbouring amino acid residues at positions 53 and 54 and 48 and 49may also be deleted.

It is to be understood that the present invention provides embodimentswhere the above modifications may be made in isolation, or in anycombination.

Removal of either the constriction at Tyr51 or the constriction atAsn55/Tyr56 results in a single constriction within the CsgG channel.Without wishing to be bound by theory, it is postulated that theconstriction at Asn55/Tyr56 would have higher conformational stabilitythan the constriction at Tyr51 which may be desirable. However, theAsn55/Tyr56 constriction could be too high (as measured along thecentral pore axis) in comparison to the nucleotides. This may lead topoor resolution of individual base pairs in translocating DNA strands.

The opposite is likely true for the Tyr51 constriction. After theremoval of the Asn55/Tyr56 constriction, the remaining ring of Tyr51residues in the oligomer may be conformationally less stable than in thenative structure. However, the Tyr51 constriction is shorter (whenmeasured along the central pore axis) and likely more capable ofproviding a constriction within the channel that may distinguish betweenindividual bases.

In either embodiment, the presence of a single narrow constriction (whenmeasured along the central pore axis) is likely to reduce the complexityof the electrical current readings when the pore is utilised in nucleicacid sequencing applications. Modulations in the observed electricalcurrent occurring during nucleic acid translocation through the porewill, hence, solely reflect the passage of separate nucleotides througha single constriction, or “reading head”.

The effective removal of one constriction may also increase theopen-channel current of the pore variant. An increased open-channelcurrent would be advantageous as a higher background conductance leadsto better resolved current blockade levels for the different nucleicacid base pair signals. In this way, modifications to the reading headcan improve the suitability of the biological pore for both nucleic acidsequencing and other molecular sensing applications.

As an alternative embodiment, or in addition to the sequencemodifications described above, it is also provided that the Asn55/Phe56constriction may be further adapted to tune its height (as measuredalong the central pore axis). Such further adaptation of the Asn55/Phe56constriction may or may not be accompanied by mutations of the Tyr51 orother positions within the CsgG channel. Suitably, further adaptation ofthe Asn55/Phe56 constriction is contemplated as part of mutations thatwiden or remove the constriction formed by the Tyr51 residue.

In the wild-type form, the Asn55/Phe56 channel constriction is composedof two amino acid rings positioned vertically adjacent to each other.The constriction, as a result, has a length of more than 1 nm. A 1 nmlong constriction may not allow the resolution of the electrical signalsgenerated from the ion flow to the separate bases in translocatingnucleic acid strands. Typically, the constriction(s) of known nanoporesused for nucleic acid sequencing typically has a length less than 1 nm.For example, the MspA nanopore which is used for DNA sequencing has aconstriction height of 0.6 nm (as measured along the central pore axis;Manrao et al., Nature Biotechnology, 2012, 30(4), 349-353).

To reduce the height of the Asn55/Phe56 constriction (as measured alongthe central pore axis), either of the two residues may be substituted ordeleted leading to a widening of the top or bottom of the constriction.

For Asn55, which forms part of the second constriction in the CsgGchannel, substitution with any amino acid is contemplated. Inparticular, substitution with alanine, glycine, valine, serine orthreonine (SEQ ID: 9-33, 44-68, 79-103 114-138, 149-173, 184-208,219-243, 254-278, 289-313 and 324-348).

For Phe 56 which forms part of the second constriction in the CsgGchannel, substitution with any amino acid is contemplated. Inparticular, substitution with alanine, glycine, valine, leucine,isoleucine, asparagine, and glutamine (SEQ ID: 5-13, 15-18, 20-23,25-28, 30-33, 40-43, 45-48, 50-53, 55-58, 60-63, 65-68, 75-78, 80-83,85-88, 90-93, 95-98, 100-103, 110-113, 115-118, 120-123, 125-128,130-133, 135-138, 145-148, 150-153, 155-158, 160-163, 165-168, 170-173,180-183, 185-188, 190-193, 195-198, 200-203, 205-208, 215-218, 220-223,225-228, 230-233, 235-238, 240-243, 250-253, 255-258, 260-263, 265-268,270-273, 275-578, 285-288, 290-293, 295-298, 300-303, 305-308, 310-313,320-323, 325-328, 330-333, 335-338, 340-343 and 345-348). In embodimentsof the invention, substitution of Phe56 with alanine and glycine isparticularly suitable (SEQ ID: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130,135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200,205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270,275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340,345, 350, 355, 360, 365, 370, 375, 380, 385, 6, 11, 16, 21, 26, 31, 36,41, 46, 51, 56, 61, 66, 71, 76, 81, 86, 91, 96, 101, 106, 111, 116, 121,126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186, 191,196, 201, 206, 211, 216, 221, 226, 231, 236, 241, 246, 251, 256, 261,266, 271, 276, 281, 286, 291, 296, 301, 306, 311, 316, 321, 326, 331,336, 341 346, 351, 356, 361, 366, 371, 376, 381 and 386).

Modifications to tune the minimum diameter of the constriction of thewild-type CsgG pore are also contemplated. The minimum diameter of bothconstrictions in the CsgG pore is approximately 0.9 nm (9 Å), which isless than the diameter of 1.2 nm for the constriction in the known MspAnanopore that shows utility for DNA sequencing (Manrao et al., NatureBiotechnology, 2012, 30(4), 349-353). Any of the above mutations abovethat provide the remaining constriction in the modified CsgG pore with aminimum diameter of 0.5 to 1.5 nm would be suitable.

Any of the modifications listed above may beneficially alter thehydrophilicity and charge distribution of the amino acids at theconstriction to improve the passage and non-covalent interaction withthe translocating nucleic acid strand in order to improve the currentread-out. Any of the mutations listed above may also beneficially alterthe hydrophilicity and charge distribution close to the channelconstriction in order to optimise the flow of electrolyte ions throughthe constriction and achieve a better discrimination among the passingnucleotides of the translocating nucleic acid strand.

Further modifications of the CsgG protein are contemplated that mayresult in changing the surface charge distribution within the channellumen. In one embodiment of the invention, these modifications may bemade to avoid undesired electrostatic adsorption of the translocatingnucleic acid to the channel wall. Since a nucleic acid is negativelycharged and the CsgG channel lumen contains some positive charges (FIG.1), it is postulated that electrostatic interaction may interfere withthe threading or translocation during nucleic acid sequencing. Suitablypositively charged amino acid residues such as lysine, histidine andarginine may be substituted with neutral or negatively charged sidechains in order to further improve the efficiency of nucleic acidtranslocation through the pore, and thus the clarity of electricalcurrent readout.

Modification of the channel lumen of wild-type E. coli CsgG or mutantCsgG to facilitate the translocation or threading of a nucleic acidstrand (or individual nucleotides) into the pore constriction is alsoprovided by an embodiment of the present invention. Themembrane-spanning section of CsgG with the inner constriction resemblesa barrel with a lid featuring a central hole. The threading could befacilitated by adding additional loops into the pore lumen which isclosest to the barrel and lid.

A specific embodiment of the invention provides that the CsgG pore maybe comprised of one or more monomers, dimers or oligomers that arecovalently attached. By way of non-limiting example, monomers may begenetically fused in any configuration, such as by their terminal aminoacids. In this instance, the amino terminus of one monomer may be fusedto the carboxy terminus of another monomer.

According to an embodiment of the invention, it is also provided thatthe CsgG pore may be adapted to accommodate additional accessoryproteins that may have beneficial properties on the passage of moleculesthrough the pore. The adaptations to the pore may facilitate anchoringof nucleic acid-processing enzymes. Nucleic acid-processing enzymes mayinclude DNA or RNA polymerases; isomerases; topoisomerases; gyrases;telomerases; and helicases. Associated of one or more of these enzymeswith the nanopore can have benefits in terms of enhanced threading ofthe nucleic acid into the pore, and in controlling the speed at which anucleic acid strand translates through the pore (Manrao et al., NatureBiotechnology, 2012, 30(4), 349-353). Controlling the translocationspeed of the nucleic acid strand through the pore has the advantage ofproviding an improved response in terms of the electrical currentmeasurement of the ion flow that is more suitable for reading and moreuniform.

In embodiments of the invention, it is envisaged that modifications inthe extra-membranous regions of the nanopore may help facilitate thedocking of a suitable nucleic acid-processing enzyme, such as aDNA-polymerase, inside or adjacent to the lumen of the channel via theprovision of one or more binding/anchoring sites. Suitable anchoringsites may comprise electrostatic patches for electrostatic binding ofthe enzyme; one or more cysteine residues to a low for covalentcoupling; and/or an altered inner width of the transmembrane channelsection to provide a steric anchor.

Further adaptations of the CsgG wild type pore for use in nucleic acidsequencing that are provided by the present invention in specificembodiments that are set out in more detail below.

In embodiments of the invention, the extra-membranous region of the CsgGpore (bottom portion as shown in FIG. 1) may be truncated or removed tofacilitate the exit of the nucleic strand on the other side of thechannel lumen. Truncation or removal of the extra-membranous region canalso improve the current resolution of the electrical signal from theion flow in the channel. The letter benefit is brought about by loweringthe resistance caused to ion flow by the extra-membranous region. In thepresent CsgG pore, the transmembrane channel, the inner constriction,and cap region represent three areas of resistance in series. Removingor lowering the contribution of one of these will increase theopen-channel current and hence improve the electrical currentresolution. Such alteration can include the deletion of α-helix 2 (α2),the C-terminal α-helix (αC) (FIG. 1), and/or a combination thereof.

In further embodiments of the invention, the membrane facing amino acidson the outer face of the wild-type E. coli CsgG pore may be modified tofacilitate the insertion of the pore into the membrane. In certainembodiments, it is provided that single amino-acid substitutions mayreplace wild-type residues with suitable more hydrophobic analogues. Forexample, one or more of the residues Ser136, Gly138, Gly140, Ala148, Ala188 or Gly202 could be changed to Ala, Val, Leu, or Ile. In addition,aromatic residues such as tyrosine or tryptophan can substituteappropriate wild-type amino-acid so that they are positioned at theinterface the hydrophobic membrane and the hydrophilic solvent. Forexample, one or more of the residues Leu154 or Leu182 could besubstituted by Tyr, Phe or Trp.

In embodiments of the invention, the thermal stability of the proteinpore may be increased. This results in an advantageous increase in theshelf-life of the nanopore in sequencing devices. In embodiments, anincrease in thermal stability of the protein pore is attained by themodification of beta-turn sequences or improving electrostaticinteractions at the protein surface. In one embodiment, the β-hairpin inthe trans-membrane regions could be stabilized by a covalent disulfideformation across two adjacent β-strands within or between adjacentβ-hairpins. Examples of such cross-strand cysteine pairs could be:Val139-Asp203; Gly139-Gly205; Lys135-Thr207; Glu201-Ala141;Gly147-Gly189; Asp149-Gln187; Gln151-Glu185; Thr207-Glu185;Gly205-Gln187; Asp203-Gly189; Ala153-Lys135; Gly137-Gln151;Val139-Asp149 or Ala141-Gly147.

In further embodiments of the invention, the codon usage of thepolypeptide sequence may be modified to allow expression of the CsgGprotein at high level and with a low error rate according to the methodsdescribed in Biotechnol. J., 2011, 6(6), 650-659. The modification mayalso target any secondary structures of the mRNA.

The present invention also provides for the alteration of the proteinsequence to improve its protease stability. This may be achieved byremoval of flexible loop regions, for example the deletion of α-helix 2(α2), the C-terminal a-helix (αC) (FIG. 1), and/or a combinationthereof.

In embodiments of the invention, the CsgG polypeptidesequence/expression system is altered to avoid the possible aggregationof the protein.

The present invention also provides for the alteration of thepolypeptide sequence to improve the folding efficiency of the protein.Suitable techniques are provided in Biotechnol. J., 2011, 6(6), 650-659.

One embodiment of the invention further provides for the replacement ofor addition of a bioaffinity tag to facilitate the purification of theCsgG protein. The published structure of the CsgG pore contains aStrepII tag (Goyal et al., Nature, 2014, 516(7530), 250-3). Embodimentsof the invention comprise other bioaffinity tags, such as Histidine-tagto facilitate the purification via metal chelate affinitychromatography. In alternative embodiments of the invention, the tag mayinclude a FLAG-tag or an epitope tag, such as a Myc- or HA-tag. In afurther embodiment of the invention, the nanopore may be modified bybiotinylation with biotin or an analogue thereof (e.g. desthiobiotin),thereby facilitating purification via interaction with streptavidin.

In embodiments of the invention, negative charges at the proteinterminus may be added to increase the net charge of the polypeptide andfacilitate the migration of the protein in polyacrylamide gelelectrophoresis. This may lead to the improved separation ofheteroligomers of the CsgG in the case where these species are ofinterest (see Howorka at al. Proc. Nat. Acad. Sci., 2001, 98(23),12996-13001). A heterooligomer can be useful to introduce asingle-cysteine residue per pore which may be advantageous infacilitating the attachment of a suitable nucleic acid-processingenzyme, such as a DNA-polymerase as described above.

The present invention relates in part to the use of the wild type ormodified CsgG nanopore in molecular sensing applications based onvariations of electrical measurements of current flowing through ananopore.

The binding of a molecule in the channel of the CsgG pore, or in thevicinity of either opening of the channel will have an effect on theopen-channel ion flow through the pore. In a similar manner to thenucleic acid sequencing application described above, variation in theopen-channel ion flow can be measured using suitable measurementtechniques by the change in electrical current (for example, WO2000/28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106,7702-7 or WO 2009/077734). The degree of reduction in ion flow, asmeasured by the reduction in electrical current, is related to the sizeof the obstruction within, or in the vicinity of, the pore. Binding of amolecule of interest, also referred to as an analyte, in or near thepore therefore provides a detectable and measurable event, therebyforming the basis of a biological sensor. Suitable molecules fornanopore sensing include nucleic acids; proteins; peptides; and smallmolecules such as pharmaceuticals, toxins or cytokines.

Detecting the presence of biological molecules finds application inpersonalised drug development, medicine, diagnostics, life scienceresearch, environmental monitoring and in the security and/or thedefence industry.

In embodiments of the invention, the wild type or modified E. coli CsgGnanopore, or homologue thereof, disclosed herein may serve as amolecular sensor. Procedures for analyte detection are described inHoworka et al. Nature Biotechnology (2012) Jun. 7; 30(6):506-7. Theanalyte molecule that is to be detected may bind to either face of thechannel, or within the lumen of the channel itself. The position ofbinding may be determined by the size of the molecule to be sensed. Thewild-type CsgG pore may act as sensor, or embodiments of the invention,the CsgG pore is modified via recombinant or chemical methods toincrease the strength of binding, the position of binding, or thespecificity of binding of the molecule to be sensed. Typicalmodifications include addition of a specific binding moietycomplimentary to the structure of the molecule to be sensed. Where theanalyte molecule comprises a nucleic acid, this binding moiety maycomprise a cyclodextrin or an oligonucleotide; for small molecules thismay be a known complimentary binding region, for example the antigenbinding portion of an antibody or of a non-antibody molecule, includinga single chain variable fragment (scFv) region or an antigen recognitiondomain from a T-cell receptor (TCR); or for proteins, it may be a knownligand of the target protein. In this way the wild type or modified E.coli CsgG nanopore, or homologue thereof, may be rendered capable ofacting as a molecular sensor for detecting presence in a sample ofsuitable antigens (including epitopes) that may include cell surfaceantigens, including receptors, markers of solid tumours or haematologiccancer cells (e.g. lymphoma or leukaemia), viral antigens, bacterialantigens, protozoal antigens, allergens, allergy related molecules,albumin (e.g. human, rodent, or bovine), fluorescent molecules(including fluorescein), blood group antigens, small molecules, drugs,enzymes, catalytic sites of enzymes or enzyme substrates, and transitionstate analogues of enzyme substrates.

Modifications may be achieved using known genetic engineering andrecombinant DNA techniques. The positioning of any adaptation would bedependent on the nature of the molecule to be sensed, for example, thesize, three-dimensional structure, and its biochemical nature. Thechoice of adapted structure may make use of computational structuraldesign. A series of bespoke CsgG nanopores is envisaged each adaptedspecifically to the sensing application to which it is destined.Determination and optimization of protein-protein interactions orprotein-small molecule interactions can be investigated usingtechnologies such as a BIAcore® which detects molecular interactionsusing surface plasmon resonance (BIAcore, Inc., Piscataway, N.J.; seealso www.biacore.com).

In an embodiment, the CsgG pore can be that of the water soluble,octameric form where the N-terminal Cys residue is replaced by analternative aminoacid in order to prevent the lipidation of the proteinN-terminus. In an alternative embodiment, the protein can be expressedin the cytoplasm by removal of the N-terminal leader sequence in orderto avoid processing by the bacterial lipidation pathway.

The method of manufacture of the CsgG monomeric soluble protein,octameric soluble protein and the oligomeric lipidated CsgG pore isdescribed in Goyal et al. (Nature, 2014; 516(7530): 250-3), which isincorporated herein by reference, and in Examples 1 and 2.

Mutant CsgG Monomers

The present invention provides mutant CsgG monomers. The mutant CsgGmonomers may be used to form the pores of the invention. A mutant CsgGmonomer is a monomer whose sequence varies from that of a wild-type CsgGmonomer and which retains the ability to form a pore. Methods forconfirming the ability of mutant monomers to form pores are well-knownin the art and are discussed in more detail below.

The mutant monomers have improved polynucleotide reading properties i.e.display improved polynucleotide capture and nucleotide discrimination.In particular, pores constructed from the mutant monomers capturenucleotides and polynucleotides more easily than the wild type. Inaddition, pores constructed from the mutant monomers display anincreased current range, which makes it easier to discriminate betweendifferent nucleotides, and a reduced variance of states, which increasesthe signal-to-noise ratio. In addition, the number of nucleotidescontributing to the current as the polynucleotide moves through poresconstructed from the mutants is decreased. This makes it easier toidentify a direct relationship between the observed current as thepolynucleotide moves through the pore and the polynucleotide sequence.In addition, pores constructed from the mutant monomers may display anincreased throughput, i.e. are more likely to interact with an analyte,such as a polynucleotide. This makes it easier to characterise analytesusing the pores. Pores constructed from the mutant monomers may insertinto a membrane more easily.

A mutant monomer of the invention comprises a variant of the sequenceshown in SEQ ID NO: 390. SEQ ID NO: 390 is the wild-type CsgG monomerfrom Escherichia coli Str. K-12 substr. MC4100. A variant of SEQ ID NO:390 is a polypeptide that has an amino acid sequence which varies fromthat of SEQ ID NO: 390 and which retains its ability to form a pore. Theability of a variant to form a pore can be assayed using any methodknown in the art. For instance, the variant may be inserted into anamphiphilic layer along with other appropriate subunits and its abilityto oligomerise to form a pore may be determined. Methods are known inthe art for inserting subunits into membranes, such as amphiphiliclayers. For example, subunits may be suspended in a purified form in asolution containing a triblock copolymer membrane such that it diffusesto the membrane and is inserted by binding to the membrane andassembling into a functional state.

In all of the discussion herein, the standard one letter codes for aminoacids are used. These are as follows: alanine (A), arginine (R),asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E),glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L),lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S),threonine (T), tryptophan (W), tyrosine (Y) and valine (V). Standardsubstitution notation is also used, i.e. Q42R means that Q at position42 is replaced with R.

In one embodiment, the mutant monomers of the invention comprise avariant of SEQ ID NO: 390 comprising one or more of the following (i)one or more mutations at the following positions (i.e. mutations at oneor more of the following positions) N40, D43, E44, S54, S57, Q62, R97,E101, E124, E131, R142, T150 and R192, such as one or more mutations atthe following positions (i.e. mutations at one or more of the followingpositions) N40, D43, E44, S54, S57, Q62, E101, E131 and T150 or N40,D43, E44, E101 and E131; (ii) mutations at Y51/N55, Y51/F56, N55/F56 orY51/N55/F56; (iii) Q42R or Q42K; (iv) K49R; (v) N102R, N102F, N102Y orN102W; (vi) D149N, D149Q or D149R; (vii) E185N, E185Q or E185R; (viii)D195N, D195Q or D195R; (ix) E201N, E201Q or E201R; (x) E203N, E203Q orE203R; and (xi) deletion of one or more of the following positions F48,K49, P50, Y51, P52, A53, S54, N55, F56 and S57. The variant may compriseany combination of (i) to (xi). In particular, the variant may comprise{i} {ii} {iii} {iv} {v} {vi} {vii} {viii} {ix} {x} {xi} {i,ii} {i,iii}{i,iv} {i,v} {i,vi} {i,vii} {i,viii} {i,ix} {i,x} {i,xi} {ii,iii}{ii,iv} {ii,v} {ii,vi} {ii,vii} {ii,viii} {ii,ix} {ii,x} {ii,xi}{iii,iv} {iii,v} {iii,vi} {iii,vii} {iii,viii} {iii,ix} {iii,x} {iii,xi}{iv,v} {iv,vi} {iv,vii} {iv,viii} {iv,ix} {iv,x} {iv,xi} {v,vi} {v,vii}{v,viii} {v,ix} {v,x} {v,xi} {vi,vii} {vi,viii} {vi,ix} {vi,x} {vi,xi}{vii,vii} {vii,ix} {vii,x} {vii,xi} {viii,ix} {viii,x} {viii,xi} {ix,x}{ix,xi} {x,xi} {i,ii,iii} {i,ii,iv} {i,ii,v} {i,ii,vi} {i,ii,vii}{i,ii,viii} {i,ii,ix} {i,ii,x} {i,ii,xi} {i,iii,iv} {i,iii,v} {i,iii,vi}{i,iii,vii} {i,iii,viii} {i,iii,ix} {i,iii,x} {i,iii,xi} {i,iv,v}{i,iv,vi} {i,iv,vii} {i,iv,vii} {i,iv,ix} {i,iv,x} {i,iv,xi} {i,v,vi}{i,v,vi} {i,v,vii} {i,v,ix} {i,v,x} {i,v,xi} {i,vi,vii} {i,vi,vii}{i,vi,ix} {i,vi,x} {i,vi,xi} {i,vii,viii} {i,vi,ix} {i,vii,x} {i,vii,xi}{i,viii,ix} {i,viii,x} {i,viii,xi} {i,ix,x} {i,ix,xi} {i,x,xi}{ii,iii,iv} {ii,ii,v} {ii,iii,vi} {ii,iii,vii} {ii,iii,viii} {ii,iii,ix}{ii,iii,x} {ii,iii,xi} {ii,iv,v} {ii,iv,vi} {ii,iv,vii} {ii,iv,viii}{ii,iv,ix} {ii,iv,x} {ii,iv,xi} {ii,v,vi} {ii,v,vii} {ii,v,viii}{ii,v,ix} {ii,v,x} {ii,v,xi} {ii,vi,vii} {ii,vi,viii} {ii,vi,ix}{ii,vi,x} {ii,vi,xi} {ii,vii,viii} {ii,vii,ix} {ii,vii,x} {ii,vii,xi}{ii,viii,ix} {ii,viii,x} {ii,viii,xi} {ii,ix,x} {ii,ix,xi} {ii,x,xi}{iii,iv,v} {iii,iv,vi} {iii,iv,vii} {iii,iv,viii} {iii,iv,ix} {iii,iv,x}{iii,iv,xi} {iii,v,vi} {iii,v,vii} {iii,v,viii} {iii,v,ix} {iii,v,x}{iii,v,xi} {iii,vi,vii} {iii,vi,viii} {iii,vi,ix} {iii,vi,x} {iii,vi,xi}{iii,vii,viii} {iii,vii,ix} {iii,vii,x} {iii,vii,xi} {iii,viii,ix}{iii,viii,x} {i,ii,iii,iv} {i,ii,iii,v} {i,ii,iii,vi} {i,ii,iii,vii}{i,ii,iii,viii} {i,ii,iii,ix} {i,ii,iii,x} {i,ii,iii,xi} {i,ii,iv,v}{i,ii,iv,vi} {i,ii,iv,vii} {i,ii,iv,viii} {i,ii,iv,ix} {i,ii,iv,x}{i,ii,iv,xi} {i,ii,v,vi} {i,ii,v,vii} {i,ii,v,viii} {i,ii,v,ix}{i,ii,v,x} {i,ii,v,xi} {i,ii,vi,vii} {i,ii,vi,viii} {i,ii,vi,ix}{i,ii,vi,x} {i,ii,vi,xi} {i,ii,vii,viii} {i,ii,vii,ix} {i,ii,vii,x}{i,ii,vii,xi} {i,ii,viii,ix} {i,ii,viii,x} {i,ii,viii,xi} {i,ii,ix,x}{i,ii,ix,xi} {i,ii,x,xi} {i,iii,iv,v} {i,iii,iv,vi} {i,iii,iv,vii}{i,iii,iv,viii} {i,iii,iv,ix} {i,iii,iv,x} {i,iii,iv,xi} {i,iii,v,vi}{i,iii,v,vii} {i,iii,v,viii} {i,iii,v,ix} {i,iii,v,x} {i,iii,v,xi}{i,iii,vi,vii} {i,iii,vi,viii} {i,iii,vi,ix} {i,iii,vi,x} {i,iii,vi,xi}{i,iii,vii,viii} {i,iii,vii,ix} {i,iii,vii,x} {i,iii,vii,xi}{i,iii,viii,ix} {i,iii,vii,x} {i,iii,viii,xi} {i,iii,ix,x} {i,iii,ix,xi}{i,iii,x,xi} {i,iv,v,vi} {i,iv,v,vii} {i,iv,v,viii} {i,iv,v,ix}{i,iv,v,x} {i,iv,v,xi} {i,iv,vi,vii} {i,iv,vi,viii} {i,iv,vi,ix}{i,iv,vi,x} {i,iv,vi,xi} {i,iv,vii,viii} {i,iv,vii,ix} {i,iv,vii,x}{i,iv,vii,xi} {i,iv,viii,ix} {i,iv,viii,x} {i,iv,viii,xi} {i,iv,ix,x}{i,iv,ix,xi} {i,iv,x,xi} {i,v,vi,vii} {i,v,vi,viii} {i,v,vi,ix}{i,v,vi,x} {i,v,vi,xi} {i,v,vii,viii} {i,v,vii,ix} {i,v,vii,x}{i,v,vii,xi} {i,v,viii,ix} {i,v,viii,x} 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{ii,iii,iv,v,vi,viii,ix,xi}{ii,iii,iv,v,vi,viii,xi} {ii,iii,iv,v,vi,ix,x,xi}{ii,iii,iv,v,vii,viii,ix,x} {ii,iii,iv,v,vii,viii,ix,xi}{ii,iii,iv,v,vii,viii,x,xi} {ii,iii,iv,v,vii,ix,x,xi}{ii,iii,iv,v,viii,ix,x,xi} {ii,iii,iv,vi,vii,viii,ix,x}{ii,iii,iv,vi,vii,viii,ix,xi} {ii,iii,iv,vi,vii,viii,x,xi}{ii,iii,iv,vi,vii,ix,x,xi} {ii,iii,iv,vi,viii,ix,x,xi}{ii,iii,iv,vii,viii,ix,x,xi} {ii,iii,v,vi,vii,viii,ix,x}{ii,iii,v,vi,vii,viii,ix,xi} {ii,iii,v,vi,vii,viii,x,xi}{ii,iii,v,vi,vii,ix,x,xi} {ii,iii,v,vi,viii,ix,x,xi}{ii,iii,v,vii,viii,ix,x,xi} {ii,iii,vi,vii,viii,ix,x,xi}{ii,iv,v,vi,vii,viii,ix,x} {ii,iv,v,vi,vii,viii,ix,xi}{ii,iv,v,vi,vii,viii,x,xi} {ii,iv,v,vi,vii,ix,x,xi}{ii,iv,v,vi,viii,ix,x,xi} {ii,iv,v,vii,viii,ix,x,xi}{ii,iv,vi,vii,viii,ix,x,xi} {ii,v,vi,vii,viii,ix,x,xi}{iii,iv,v,vi,vii,viii,ix,x} {iii,iv,v,vi,vii,viii,ix,xi}{iii,iv,v,vi,vii,viii,x,xi} {iii,iv,v,vi,vii,ix,x,xi}{iii,iv,v,vi,viii,ix,x,xi} {iii,iv,v,vii,viii,ix,x,xi}{iii,iv,vi,vii,viii,ix,x,xi} {iii,v,vi,vii,viii,ix,x,xi}{iv,v,vi,vii,viii,ix,x,xi} {i,ii,iii,iv,v,vi,vii,viii,ix}{i,ii,iii,iv,v,vi,vii,viii,x} {i,ii,iii,iv,v,vi,vii,viii,xi}{i,ii,iii,iv,v,vi,vii,ix,x} {i,ii,iii,iv,v,vi,vii,ix,xi}{i,ii,iii,iv,v,vi,vii,x,xi} {i,ii,iii,iv,v,vi,viii,ix,x}{i,ii,iii,iv,v,vi,viii,ix,xi} {i,ii,iii,iv,v,vi,viii,x,xi}{i,ii,iii,iv,v,vi,ix,x,xi} {i,ii,iii,iv,v,vii,viii,ix,x}{i,ii,iii,iv,v,vii,viii,ix,xi} {i,ii,iii,iv,v,vii,viii,x,xi}{i,ii,iii,iv,v,vii,ix,x,xi} {i,ii,iii,iv,v,viii,ix,x,xi}{i,ii,iii,iv,vi,vii,viii,ix,x} {i,ii,iii,iv,vi,vii,viii,ix,xi}{i,ii,iii,iv,vi,vii,viii,x,xi} {i,ii,iii,iv,vi,vii,ix,x,xi}{i,ii,iii,iv,vi,viii,ix,x,xi} {i,ii,iii,iv,vii,viii,ix,xi}{i,ii,iii,v,vi,vii,viii,ix,x} {i,ii,iii,v,vi,vii,viii,ix,xi}{i,ii,iii,v,vi,vii,viii,x,xi} {i,ii,iii,v,vi,vii,ix,x,xi}{i,ii,iii,v,vi,viii,ix,x,xi} {i,ii,iii,v,vii,viii,ix,x,xi}{i,ii,iii,vi,vii,viii,ix,x,xi} {i,ii,iv,v,vi,vii,viii,ix,x}{i,ii,iv,v,vi,vii,viii,ix,xi} {i,ii,iv,v,vi,vii,viii,x,xi}{i,ii,iv,v,vi,vii,ix,x,xi} {i,ii,iv,v,vi,viii,ix,x,xi}{i,ii,iv,v,vii,viii,ix,x,xi} {i,ii,iv,vi,vii,viii,ix,x,xi}{i,ii,v,vi,vii,viii,ix,x,xi} {i,iii,iv,v,vi,vii,viii,ix,x}{i,iii,iv,v,vi,vii,viii,ix,xi} {i,iii,iv,v,vi,vii,viii,x,xi}{i,iii,iv,v,vi,vii,ix,x,xi} {i,iii,iv,v,vi,viii,ix,x,xi}{i,iii,iv,v,vii,viii,ix,x,xi} {i,iii,iv,vi,vii,viii,ix,x,xi}{i,iii,v,vi,vii,viii,ix,x,xi} {i,iv,v,vi,vii,viii,ix,x,xi}{ii,iii,iv,v,vi,vii,viii,ix} {ii,iii,iv,v,vi,vii,viii,ix,xi}{ii,iii,iv,v,vi,vii,viii,xi} {ii,iii,iv,v,vi,vii,ix,xi}{ii,iii,iv,v,vi,viii,ix,x,xi} {ii,iii,iv,v,vii,viii,ix,x,xi}{ii,iii,iv,vi,vii,viii,ix,x,xi} {ii,iii,v,vi,vii,viii,ix,x,xi}{ii,iv,v,vi,vii,viii,ix,x,xi} {iii,iv,v,vi,vii,viii,ix,x,xi}{i,ii,iii,iv,v,vi,vii,viii,ix,x} {i,ii,iii,iv,v,vi,vii,viii,ix,xi}{i,ii,iii,iv,v,vi,vii,viii,x,xi} {i,ii,iii,iv,v,vi,vii,ix,x,xi}{i,ii,iii,iv,v,vi,viii,ix,x,xi} {i,ii,iii,iv,v,vii,viii,ix,x,xi}{i,ii,iii,iv,vi,vii,viii,ix,x,xi} {i,ii,iii,v,vi,vii,viii,ix,x,xi}{i,ii,iv,v,vi,vii,viii,ix,x,xi} {i,iii,iv,v,vi,vii,viii,ix,x,xi}{ii,iii,iv,v,vi,vii,viii,ix,x,xi} or{i,ii,iii,iv,v,vi,vii,viii,ix,x,xi}.

If the variant comprises any one of (i) and (iii) to (xi), it mayfurther comprise a mutation at one or more of Y51, N55 and F56, such asat Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56.

In (i), the variant may comprises mutations at any number andcombination of N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131,R142, T150 and R192. In (i), the variant preferably comprises one ormore mutations at the following positions (i.e. mutations at one or moreof the following positions) N40, D43, E44, S54, S57, Q62, E101, E131 andT150. In (i), the variant preferably comprises one or more mutations atthe following positions (i.e. mutations at one or more of the followingpositions) N40, D43, E44, E101 and E131. In (i), the variant preferablycomprises a mutation at S54 and/or S57. In (i), the variant morepreferably comprises a mutation at (a) S54 and/or S57 and (b) one ormore of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56,N55/F56 or Y51/N55/F56. If S54 and/or S57 are deleted in (xi), it/theycannot be mutated i (i) and vice versa. In (i), the variant preferablycomprises a mutation at T150, such as T150I. Alternatively the variantpreferably comprises a mutation at (a) T150 and (b) one or more of Y51,N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 orY51/N55/F56. In (i), the variant preferably comprises a mutation at Q62,such as Q62R or Q62K. Alternatively the variant preferably comprises amutation at (a) Q62 and (b) one or more of Y51, N55 and F56, such as atY51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56. The variant maycomprise a mutation at D43, E44, Q62 or any combination thereof, such asD43, E44, Q62, D43/E44, D43/Q62, E44/Q62 or D43/E44/Q62. Alternativelythe variant preferably comprises a mutation at (a) D43, E44, Q62,D43/E44, D43/Q62, E44/Q62 or D43/E44/Q62 and (b) one or more of Y51, N55and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 orY51/N55/F56.

In (ii) and elsewhere in this application, the/symbol means “and” suchthat Y51/N55 is Y51 and N55. In (ii), the variant preferably comprisesmutations at Y51/N55. It has been proposed that the constriction in CsgGis composed of three stacked concentric rings formed by the side chainsof residues Y51, N55 and F56 (Goyal et al, 2014, Nature, 516, 250-253).Mutation of these residues in (ii) may therefore decrease the number ofnucleotides contributing to the current as the polynucleotide movesthrough the pore and thereby make it easier to identify a directrelationship between the observed current (as the polynucleotide movesthrough the pore) and the polynucleotide. Y56 may be mutated in any ofthe ways discussed below with reference to variants and pores useful inthe method of the invention.

In (v), the variant may comprise N102R, N102F, N102Y or N102W. Thevariant preferably comprises (a) N102R, N102F, N102Y or N102W and (b) amutation at one or more of Y51, N55 and F56, such as at Y51, N55, F56,Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56.

In (xi), any number and combination of K49, P50, Y51, P52, A53, S54,N55, F56 and S57 may be deleted. Preferably one or more of K49, P50,Y51, P52, A53, S54, N55 and S57 may be deleted. If any of Y51, N55 andF56 are deleted in (xi), it/they cannot be mutated in (i) and viceversa.

In (i), the variant preferably comprises one of more of the followingsubstitutions N40R, N40K, D43N, D43Q, D43R, D43K, E44N, E44Q, E44R,E44K, S54P, S57P, Q62R, Q62K, R97N, R97G, R97L, E101N, E101Q, E101R,E101K, E101F, E101Y, E101W, E124N, E124Q, E124R, E124K, E124F, E124Y,E124W, E131D, R142E, R142N, T150I, R192E and R192N, such as one or moreof N40R, N40K, D43N, D43Q, D43R, D43K, E44N, E44Q, E44R, E44K, S54P,S57P, Q62R, Q62K, E101N, E101Q, E101R, E101K, E101F, E101Y, E101W, E131Dand T150I, or one or more of N40R, N40K, D43N, D43Q, D43R, D43K, E44N,E44Q, E44R, E44K, E101N, E101Q, E101R, E101K, E101F, E101Y, E101W andE131D. The variant may comprise any number and combination of thesesubstitutions. In (i), the variant preferably comprises S54P and/orS57P. In (i), the variant preferably comprises (a) S54P and/or S57P and(b) a mutation at one or more of Y51, N55 and F56, such as at Y51, N55,F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56. The mutations at one ormore of Y51, N55 and F56 may be any of those discussed below. In (i),the variant preferably comprises F56A/S57P or S54P/F56A. The variantpreferably comprises T150I. Alternatively the variant preferablycomprises a mutation at (a) T150I and (b) one or more of Y51, N55 andF56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56.

In (i), the variant preferably comprises Q62R or Q62K. Alternatively thevariant preferably comprises (a) Q62R or Q62K and (b) a mutation at oneor more of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56,N55/F56 or Y51/N55/F56. The variant may comprise D43N, E44N, Q62R orQ62K or any combination thereof, such as D43N, E44N, Q62R, Q62K,D43N/E44N, D43N/Q62R, D43N/Q62K, E44N/Q62R, E44N/Q62K, D43N/E44N/Q62R orD43N/E44N/Q62K. Alternatively the variant preferably comprises (a) D43N,E44N, Q62R, Q62K, D43N/E44N, D43W/Q62R, D43N/Q62K, E44N/Q62R, E44N/Q62K,D43N/E44N/Q62R or D43N/E44N/Q62K and (b) a mutation at one or more ofY51, N55 and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 orY51/N55/F56.

In (i), the variant preferably comprises D43N.

In (i), the variant preferably comprises E101R, E101S, E101F or E101N.

In (i), the variant preferably comprises E124N, E124Q, E124R, E124K,E124F, E124Y, E124W or E124D, such as E124N.

In (i), the variant preferably comprises R142E and R142N.

In (i), the variant preferably comprises R97N, R97G or R97L.

In (i), the variant preferably comprises R192E and R192N.

In (ii), the variant preferably comprises F56N/N55Q, F56N/N55R,F56N/N55K, F56N/N55S, F56N/N55G, F56N/N55A, F56N/N55T, F56Q/N55Q,F56Q/N55R, F56Q/N55K, F56Q/N55S, F56Q/N55G, F56Q/N55A, F56Q/N55T,F56R/N55Q, F56R/N55R, F56R/N55K, F56R/N55S, F56R/N55G, F56R/N55A,F56R/N55T, F56S/N55Q, F56S/N55R, F58S/N55K, F56S/N55S, F56S/N55G,F56S/N55A, F56S/N55T, F56G/N55Q, F56G/N55R, F56G/N55K, F56G/N55S,F56G/N55G, F56G/N55A, F56G/N55T, F56A/N550, F56A/N55R, F56A/N55K,F56A/N55S, F56A/N55G, F56A/N55A, F56A/N55T, F56K/N55Q, F56K/N55R,F56K/N55K, F56K/N55S, F56K/N55G, F56K/N55A, F56K/N55T, F56N/Y51L,F56N/Y51V, F56N/Y51A, F56N/Y51N, F56N/Y51Q, F56N/Y51S, F56N/Y51G,F56Q/Y51L, F56Q/Y51V, F56Q/Y51A, F56Q/Y51N, F56Q/Y51Q, F56Q/Y51S,F56Q/Y51G, F56R/Y51L, F56R/Y51V, F56R/Y51A, F56R/Y51N, F56R/Y51Q,F56R/Y51S, F56R/Y51G, F56S/Y51L, F56S/Y51V, F56/Y51A, F56S/Y51N,F56S/Y51Q, F56S/Y51S, F56S/Y51G, F56G/Y51L, F56G/Y51V, F56G/Y51A,F56G/Y51N, F56G/Y51Q, F56G/Y51S, F56G/Y51G, F56A/Y51L, F56A/Y51V,F56A/Y51A, F56A/Y51N, F56A/Y51Q, F56A/Y51S, F56A/Y51G, F56K/Y51L,F56K/Y51V, F56K/Y51A, F56K/Y51N, F56K/Y51Q, F56K/Y51S, F56K/Y51G,N55Q/Y51L, N55Q/Y51V, N55Q/Y51A, N55Q/Y51N, N55Q/Y51Q, N55Q/Y51S,N55Q/Y51G, N55R/Y51L, N55R/Y51V, N55R/Y51A, N55R/Y51N, N55R/Y51Q,N55R/Y51S, N55R/Y51G, N55K/Y51L, N55K/Y51V, N55K/Y51A, N55K/Y51N,N55K/Y51Q, N55K/Y51S, N55K/Y51G, N55S/Y51L, N55S/Y51V, N55S/Y51A,N55S/Y51N, N55S/Y51Q, N55S/Y51S, N55S/Y51G, N55G/Y51L, N55G/Y5I V,N55G/Y51A, N55G/Y51N, N55G/Y51Q, N55G/Y51S, N55G/Y51G, N55A/Y51L,N55A/Y51V, N55A/Y51A, N55A/Y51N, N55A/Y51Q, N55A/Y51S, N55A/Y51G,N55T/Y51L, N55T/Y51V, N55T/Y51A, N55T/Y51N, N55T/Y51Q, N55T/Y51S,N55T/Y51G, F56N/N55Q/Y51L, F56N/N550/Y51V, F56N/N55Q/Y51A,F56N/N55Q/Y51N, F56N/N55Q/Y51Q, F56N/N55Q/Y51S, F56N/N55Q/Y51G,F56N/N55R/Y51L, F56N/N55R/Y51V, F56N/N55R/Y51A, F56N/N55R/Y51N,F56N/N55R/Y51Q, F56N/N55R/Y51S, F56N/N55R/Y51G, F56N/N55K/Y51L,F56N/N55K/Y51V, F56N/N55K/Y51A, F56N/N55K/Y51N, F56N/N55K/Y51Q,F56N/N55K/Y51S, F56N/N55K/Y51G, F56N/N55S/Y51L, F56N/N55S/Y51V,F56N/N55S/Y51A, F56N/N55S/Y51N, F56N/N55S/Y51Q, F56N/N55S/Y51S,F56N/N55S/Y51G, F56N/N55G/Y51L, F56N/N55G/Y51V, F56N/N55G/Y51A,F56N/N55G/Y51N, F56N/N55G/Y51Q, F56N/N55G/Y51S, F56N/N55G/Y51G,F56N/N55A/Y51L, F56W/N55A/Y51V, F56N/N55A/Y51A, F56W/N55A/Y51N,F56N/N55A/Y51Q, F56N/N55A/Y51S, F56N/N55A/Y51G, F56N/N55T/Y51L,F56N/N55T/Y51V, F56N/N55T/Y51A, F56N/N55T/Y51N, F56N/N55T/Y51Q,F56N/N55T/Y51S, F56N/N55T/Y51G, F56Q/N55Q/Y51L, F56Q/N55Q/Y51V,F56Q/N55Q/Y51A, F56Q/N55Q/Y51N, F56Q/N55Q/Y51Q, F56Q/N55Q/Y51S,F56Q/N55Q/Y51G, F56Q/N55R/Y51L, F56Q/N55R/Y51V, F56Q/N55R/Y51A,F56Q/N55R/Y51N, F56Q/N55R/Y51Q, F56Q/N55R/YV51S, F56Q/N55R/Y51G,F56Q/M55K/Y51L, F56Q/N55K/Y51V, F56Q/N55K/Y51A, F56Q/N55K/Y51N,F56Q/N55K/Y51Q, F56Q/N55K/Y51S, F56Q/N55K/Y51G, F56Q/N55S/Y51L,F56Q/N55S/Y51V, F56Q/N55S/Y51A, F56Q/N55S/Y51N, F56Q/N55S/Y51Q,F56Q/N55S/Y51S, F56Q/N55S/Y51G, F56Q/N55G/Y51L, F56G/Y55G/Y51V,F56Q/N55G/Y51A, F56Q/N55G/Y51N, F56Q/N55G/Y51Q, F56Q/N55G/Y51S,F56Q/N55G/Y51G, F56Q/N55A/Y51L, F56Q/N55A/Y51V, F56Q/N55A/Y51A,F56Q/N55A/Y51N, F56Q/N55A/Y51Q, F56Q/N55A/Y51S, F56Q/N55A/Y51G,F56Q/N55T/Y51L, F56Q/N55T/Y51V, F56Q/N55T/Y51A, F56Q/N55T/Y51N,F56Q/N55T/Y51Q, F56Q/N55T/Y51S, F56Q/N55T/Y51G, F56R/N55Q/Y51L,F56R/N55Q/Y51V, F56R/N55Q/Y51A, F56R/N55Q/N51N, F56R/N55Q/Y51Q,F56R/N55Q/Y51S, F56R/N55Q/Y51G, F56R/N55R/Y51L, F56R/N55R/Y51V,F56R/N55R/Y51A, F56R/N55R/Y51N, F56R/N55R/Y51Q, F56R/N55R/Y51S,F56R/N55R/Y51G, F56R/N55K/Y51L, F56R/N55K/Y51V, F56R/N55K/Y51A,F56R/N55K/Y51N, F56R/N55K/Y51Q, F56R/N55K/Y51S, F56R/N55K/Y51G,F56R/N55S/Y51L, F56R/N55S/Y51V, F56R/N55S/Y51A, F56R/N55S/Y51N,F56R/N55S/Y51Q, F56R/N55S/Y51S, F56R/N55S/Y51Q, F56R/N55G/Y51L,F56R/N55G/Y51V, F56R/N55G/Y51A, F56R/N55G/Y51N, F56R/N55G/Y51Q,F56R/N55G/Y51S, F56R/N55G/Y51G, F56R/N55A/Y51L, F56R/N55A/Y51V,F56R/N55A/Y51A, F56R/N55A/Y51N, F56R/N55A/Y51Q, F56R/N55A/Y51S,F56R/N55A/Y51G, F56R/N55T/Y51L, F56R/N55T/Y51V, F56R/N55T/Y51A,F56R/N55T/Y51N, F56R/N55T/Y51Q, F56R/N55T/Y51S, F56R/N55T/Y51G,F56S/N55Q/N51L, F56S/N55Q/Y51V, F56S/N55Q/Y51A, F56S/N55Q/Y51N,F56S/N55Q/N51Q, F56S/N55Q/Y51S, F56S/N55Q/Y51G, F56S/N55R/Y51L,F56S/N55R/Y51V, F56S/N55R/Y51A, F56S/N55R/Y51N, F56S/N55R/Y51Q,F56S/N55R/Y51S, F56S/N55R/Y51G, F56S/N55K/Y51L, F56S/N55K/Y51V,F56S/N55K/Y51A, F56S/N55K/Y51N, F56S/N55K/Y51Q, F56S/N55K/Y51S,F56S/N55K/Y51G, F56S/N55S/Y51L, F56S/N55S/Y51V, F56S/N55S/Y51A,F56S/N55S/Y51N, F56S/N55S/Y51Q, F56S/N55S/N51S, F56S/N55S/Y51G,F56S/N55G/Y51L, F56S/N55G/Y51V, F56S/N55G/Y51A, F56S/N55G/Y51N,F56S/N55G/Y51Q, F56S/N55G/Y51S, F56S/N55G/Y51G, F56S/N55A/Y51L,F56S/N55A/Y51V, F56S/N55A/Y51A, F56S/N55A/Y51N, F56S/N55A/Y51Q,F56S/N55A/Y51S, F58S/N55A/Y51Q, F56S/N55T/Y51L, F56S/N55T/Y51V,F56S/N55T/Y51A, F56S/N55T/Y51N, F56S/N55T/Y51Q, F56S/N55T/Y51S,F56S/N55T/Y51Q, F56G/N55Q/Y51L, F56G/N55Q/Y51V, F56G/N55Q/Y51A,F56G/N55Q/Y51N, F56G/N55Q/Y51Q, F56G/N55Q/Y51S, F56G/N55Q/Y51G,F56G/N55R/Y51L, F56G/N55R/Y51V, F56G/N55R/Y51A, F56G/N55R/Y51N,F56G/N55R/Y51Q, F56G/N55R/Y51S, F56G/N55R/Y51G, F56G/N55K/Y51L,F56G/N55K/Y51V, F56Q/N55K/Y51A, F56G/N55K/Y51N, F56D/N55K/Y51Q,F56/N55K/Y51S, F56G/N55K/Y51Q, F56G/N55S/Y51L, F56G/N55S/Y51V,F56Q/N55S/Y51A, F56G/N55S/Y51N, F56G/N55S/Y51Q, F56G/N55S/Y51S,F56G/N55S/Y51G, F56G/N55G/Y51L, F56G/N55G/N51V, F56G/N55G/Y51A,F56G/N55G/Y51N, F56/N55G/Y51Q, F56G/N55G/Y51S, F56G/N55G/Y51G,F56G/N55A/Y51L, F56G/N55N51V, F56G/N55A/Y51A, F56G/N55A/Y51N,F56G/N55A/Y51Q, F56G/N55A/Y51S, F56G/N55A/Y51G, F56G/N55T/Y51L,F56G/N55T/Y51V, F56G/N55T/Y51A, F56G/N55T/Y51N, F56G/N55T/Y51Q,F56G/N55T/Y51S, F56G/N55T/Y51G, F56A/N55Q/Y51L, F56A/N55Q/Y51V,F56A/N55Q/Y51A, F56A/N55Q/Y51N, F56A/N55Q/Y51Q, F56N55Q/Y51S,F56A/N55Q/Y51G, F56A/N55R/Y51L, F56A/N55R/Y51V, F56A/N55R/Y51A,F56A/N55R/Y51N, F56A/N55R/Y51Q, F56A/N55R/Y51S, F56A/N55R/Y51G,F56A/N55K/Y51L, F56A/N55K/Y51V, F56A/N55K/Y51A, F56A/N55K/Y51N,F56A/N55K/Y51Q, F56A/N55K/Y51S, F56A/N55K/Y51G, F56A/N55S/Y51L,F56A/N55S/Y51V, F56A/N55S/Y51A, F56N/N55S/Y51N, F56A/N55S/Y51Q,F56A/N55S/Y51S, F56A/N55S/Y51G, F56A/N55G/Y51L, F56A/N55G/Y51V,F56A/N55G/Y51A, F56A/N55G/Y51N, F56A/N55G/Y51Q, F56A/N55G/Y51S,F56A/N55G/Y51G, F56A/N55A/Y51L, F56A/N55A/Y51V, F56A/N55A/Y51A,F56A/N55A/Y51N, F56A/N55A/Y51Q, F56A/N55A/Y51S, F56A/N55N/Y51G,F56A/N55T/Y51L, F56A/N55T/Y51V, F56A/N55T/Y51A, F56A/N55T/Y51N,F56A/N55T/Y51Q, F56A/N55T/Y51S, F56A/N55T/Y51G, F56K/N55Q/Y51L,F56K/N55Q/Y51V, F56K/N55Q/Y51A, F56K/N55Q/Y51N, F56K/N55Q/Y51Q,F56K/N55Q/Y51S, F56K/N55Q/Y51G, F56K/N55R/Y51L, F56K/N55R/Y51V,F56K/N55R/Y51A, F56K/N55R/Y51N, F56K/N55R/Y51Q, F56K/N55R/Y51S,F56K/N55R/Y51G, F56K/N55K/Y51L, F56K/N55K/Y51V, F56K/N55K/Y51A,F56K/N55K/Y51N, F56K/N55K/Y51Q, F56K/N55K/Y51S, F56K/N55K/Y51G,F56K/N55S/Y51L, F56K/N55S/Y51V, F56K/N55S/Y51A, F56K/N55S/Y51N,F56K/N55S/Y51Q, F56K/N55S/Y51S, F56K/N55S/Y51G, F56K/N55G/Y51L,F56K/N55G/Y51V, F56K/N55G/Y51A, F56K/N55G/Y51N, F56K/N55G/Y51Q,F56K/N55G/Y51S, F56K/N55G/Y51G, F56K/N55A/Y51L, F56K/N55A/Y51V,F56K/N55A/Y51A, F56K/N55A/Y51N, F56K/N55A/Y51Q, F56K/N55A/Y51S,F56K/N55A/Y51G, F56K/N55T/Y51L, F56K/N55T/Y51V, F56K/N55T/Y51A,F56K/N55T/Y51N, F56K/N55T/Y51Q, F56K/N55T/Y51S, F56K/N55T/Y51G,F56E/N55R, F56E/N55K, F56D/N55R, F56D/N55K, F56R/N55E, F56R/N55D,F56K/N55E or F56K/N55D.

In (ii), the variant preferably comprises Y51R/F56Q, Y51N/F56N,Y51M/F56Q, Y51L/F56Q, Y51I/F56Q, Y51V/F56Q, Y51A/F56Q, Y51P/F56Q,Y51G/F56Q, Y51C/F56Q, Y51Q/F56Q, Y51N/F56Q, Y51S/F56Q, Y51E/F56Q,Y51D/F56Q, Y51K/F56Q or Y51H/F56Q.

In (ii), the variant preferably comprises Y51T/F56Q, Y51Q/F56Q orY51A/F560.

In (ii), the variant preferably comprises Y51T/F56F, Y51T/F56M,Y51T/F56L, Y51T/F56I, Y51T/F56V, Y51T/F56A, Y51T/F56P, Y51T/F56G,Y51T/F56C, Y51T/F56Q, Y51T/F56N, Y51T/F56T, Y51T/F56S, Y51T/F56E,Y51T/F56D, Y51T/F56K, Y51T/F56H or Y51T/F56R.

In (ii), the variant preferably comprises Y51T/N55Q, Y51T/N55S orY51T/N55A.

In (ii), the variant preferably comprises Y51A/F56F, Y51A/F56L,Y51A/F56I, Y51A/F56V, Y51A/F56A, Y51A/F56P, Y51A/F56G, Y51A/F56C,Y51A/F56Q, Y51A/F56N, Y51A/F56T, Y51A/F56S, Y51A/F56E, Y51A/F56D,Y51A/F56K, Y51A/F56H or Y51A/F56R.

In (ii), the variant preferably comprises Y51C/F56A, Y51E/F56A,Y51D/F56A, Y51K/F56A, Y51H/F56A, Y51Q/F56A, Y51N/F56A, Y51S/F56A,Y51P/F56A or Y51V/F56A.

In (xi), the variant preferably comprises deletion of Y51/P52,Y51/P52/A53, P50 to P52, P50 to A53, K49 to Y51, K49 to A53 andreplacement with a single praline (P), K49 to S54 and replacement with asingle P, Y51 to A53, Y51 to S54, N55/F56, N55 to S57, N55/F56 andreplacement with a single P, N55/F56 and replacement with a singleglycine (G), N55/F56 and replacement with a single alanine (A), N55/F56and replacement with a single P and Y51N. N55/F56 and replacement with asingle P and Y51Q, N55/F58 and replacement with a single P and Y51S,N55/F56 and replacement with a single G and Y51N, N55/F56 andreplacement with a single G and Y51Q, N55/F56 and replacement with asingle G and Y51S, N55/F56 and replacement with a single A and Y51N,N55/F56 and replacement with a single A/Y51Q or N55/F56 and replacementwith a single A and Y51S.

The variant more preferably comprises D195N/E203N, D195Q/E203N,D195N/E203Q, D195Q/E203Q, E201N/E203N, E201Q/E203N, E201N/E203Q,E201Q/E203Q, E185N/E203Q, E185Q/E203Q, E185N/E203N, E185Q/E203N,D195N/E201N/E203N, D195Q/E201N/E203N, D195N/E201Q/E203N,D195N/E201N/E203Q, D195Q/E201Q/E203N, D195Q/E201N/E203Q,D195N/E201Q/E203Q, D195Q/E201Q/E203Q, D149N/E201N, D149Q/E201N,D149N/E201Q, D149Q/E201Q, D149N/E201N/D195N, D149Q/E201N/D195N,D149N/E201Q/D195N, D149N/E201N/D195Q, D149Q/E201Q/D195N,D149Q/E201N/D195Q, D149N/E201Q/D195Q, D149Q/E201Q/D195Q, D149N/E203N,D149Q/E203N, D149N/E203Q, D149Q/E203Q, D149N/E185N/E201N,D149Q/E185N/E201N, D149N/E185Q/E201N, D149N/E185N/E201Q,D149Q/E185Q/E201N, D149Q/E185N/E201Q, D149N/E185Q/E201Q,D149Q/E185Q/E201Q, D149N/E185N/E203N, D149Q/E185N/E203N,D149N/E185Q/E203N, D149N/E185N/E203Q, D149Q/E185Q/E203N,D149Q/E185N/E203Q, D149N/E185Q/E203Q, D149Q/E185Q/E203Q,D149N/E185N/E201N/E203N, D149Q/E185N/E201N/E203N,D149N/E185Q/E201N/E203N, D149N/E185N/E201Q/E203N,D149N/E185N/E201N/E203Q, D149Q/E185Q/E201N/E203N,D149Q/E185N/E201Q/E203N, D149Q/E185N/E201N/E203Q,D149N/E185Q/E201Q/E203N, D149Q/E185Q/E201N/E203Q,D149N/E185N/E201Q/E203Q, D149Q/E185Q/E201Q/E203Q,D149Q/E185Q/E201N/E203Q, D149Q/E185N/E201Q/E203Q,D149N/E185Q/E201Q/E203Q, D149Q/E185Q/E201Q/E203N,D149N/E185N/D195N/E201N/E203N, D149Q/E185N/D195N/E201N/E203N,D149N/E185Q/D195N/E201N/E203N, D149N/E185N/D195Q/E201N/E203N,D149N/E185N/D195N/E201Q/E203N, D149N/E185W/D195N/E201N/E203Q,D149Q/E185Q/D195N/E201N/E203N, D149Q/E185W/D195Q/E201N/E203N,D149Q/E185N/D195N/E201 Q/E203N, D149Q/E185N/D195N/E201N/E203Q,D149N/E185Q/D195Q/E201N/E203N, D149N/E185Q/D195N/E201Q/E203N,D149N/E185Q/D195N/E201N/E203Q, D149N/E185N/D195Q/E201Q/E203N,D149N/E185N/D195Q/E201N/E203Q, D149N/E185N/D195N/E201Q/E203Q,D149Q/E185Q/D195Q/E201N/E203N, D149Q/E185Q/D195N/E201Q/E203N,D149Q/E185Q/D195N/E201N/E203Q, D149Q/E185N/D195Q/E201Q/E203N,D149Q/E185N/D195Q/E201N/E203Q, D149Q/E185N/D195N/E201Q/E203Q,D149N/E185Q/D195Q/E201Q/E203N, D149N/E185Q/D195Q/E201N/E203Q,D149Q/E185Q/D195N/E201Q/E203Q, D149Q/E185N/D195Q/E201Q/E203Q,D149Q/E185Q/D195Q/E201Q/E203N, D149Q/E185Q/D195Q/E201N/E203Q,D149Q/E185Q/D195N/E201Q/E203Q, D149Q/E185W/D195Q/E201Q/E203Q,D149N/E185Q/D195Q/E201Q/E203Q, D149Q/E185Q/D195Q/E201Q/E203Q,D149N/E185R/E201N/E203N, D149Q/E185R/E201N/E203N,D149N/E185R/E201Q/E203N, D149N/E185R/E201N/E203Q,D149Q/E185R/E201Q/E203N, D149Q/E185R/E201N/E203Q,D149N/E185R/E201Q/E203Q, D149Q/E185R/E201Q/E203Q,D149R/E185N/E201N/E203N, D149R/E185Q/E201N/E203N,D149R/E185N/E201Q/E203N, D149R/E185N/E201N/E203Q,D149R/E185Q/E201Q/E203N, D149R/E185Q/E201N/E203Q,D149R/E185N/E201Q/E203Q, D149R/E185Q/E201Q/E203Q,D149R/E185N/D195N/E201N/E203N, D149R/E185Q/D195N/E201N/E203N,D149R/E185N/D195Q/E201N/E203N, D149R/E185N/D195N/E201Q/E203N,D149R/E185Q/D195N/E201N/E203Q, D149R/E185Q/D195Q/E201N/E203N,D149R/E185Q/D195N/E201Q/E203N, D149R/E185Q/D195N/E201N/E203Q,D149R/E185N/D195Q/E201Q/E203N, D149R/E185N/D195Q/E201N/E203Q,D149R/E185N/D195N/E201Q/E203Q, D149R/E185Q/D195Q/E201Q/E203N,D149R/E185Q/D195Q/E201N/E203Q, D149R/E185Q/D195N/E201Q/E203Q,D149R/E185N/D195Q/E201Q/E203Q, D149R/E185Q/D195Q/E201Q/E203Q,D149N/E185R/D195N/E201N/E203N, D149Q/E185R/D195N/E201N/E203N,D149N/E185R/D195Q/E201N/E203N, D149N/E185R/D195N/E201Q/E203N,D149N/E185R/D195N/E201N/E203Q, D149Q/E185R/D195Q/E201N/E203N,D149Q/E185R/D195N/E201Q/E203N, D149Q/E185R/D195N/E201N/E203Q,D149N/E185R/D195Q/E201Q/E203N, D149N/E185R/D195Q/E201N/E203Q,D149N/E185R/D195N/E201Q/E203Q, D149Q/E185R/D195Q/E201Q/E203N,D149Q/E185R/D195Q/E201N/E203Q, D149Q/E185R/D195N/E201Q/E203Q,D149N/E185R/D195Q/E201Q/E203Q, D149Q/E185R/D195Q/E201Q/E203Q,D149N/E185R/D195N/E201R/E203N, D149Q/E185R/D195N/E201R/E203N,D149N/E185R/D195Q/E201R/E203N, D149N/E185R/D195N/E201R/E203Q,D149Q/E185R/D195Q/E201R/E203N, D149Q/E185R/D195N/E201R/E203Q,D149/E185R/D195/E201R/E203Q, D149Q/E185R/D195Q/E201R/E203Q, E131D/K49R,E101N/N102F, E101N/N102Y, E101N/N102W, E101F/N102F, E101F/N102Y,E101F/N102W, E101Y/N102F, E101Y/N102Y, E101Y/N102W, E101W/N102F,E101W/N102Y, E101W/N102W, E101N/N102R, E101F/N102R, E101Y/N102R orE101W/N102F.

Preferred variants of the invention which form pores in which fewernucleotides contribute to the current as the polynucleotide movesthrough the pore comprise Y51A/F56A, Y51A/F56N, Y51I/F56A, Y51L/F56A,Y51T/F56A, Y51I/F56N, Y51L/F56N or Y51T/F56N or more preferablyY51I/F56A, Y51L/F56A or Y51T/F56A. As discussed above, this makes iteasier to identify a direct relationship between the observed current(as the polynucleotide moves through the pore) and the polynucleotide.

Preferred variants which form pores displaying an increased rangecomprise mutations at the following positions:

-   -   Y51, F56, D149, E185, E201 and E203;    -   N55 and F56;    -   Y51 and F56;    -   Y51, N55 and F56; or    -   F56 and N102.

Preferred variants which form pores displaying an increased rangecomprise:

-   -   Y51N, F56A, D149N, E185R, E201N and E203N;    -   N55S and F56Q;    -   Y51A and F56A;    -   Y51A and F56N;    -   Y51I and F56A;    -   Y51L and F56A;    -   Y51T and F56A;    -   Y51I and F56N;    -   Y51L and F56N;    -   Y51T and F56N;    -   Y51T and F56Q;    -   Y51A, N55S and F56A;    -   Y51A, N55S and F56N;    -   Y51T, N55S and F56Q; or    -   F56Q and N102R.

Preferred variants which form pores in which fewer nucleotidescontribute to the current as the polynucleotide moves through the porecomprise mutations at the following positions:

-   -   N55 and F56, such as N55X and F56Q, wherein X is any amino acid;        or    -   Y51 and F56, such as Y51X and F56Q, wherein X is any amino acid.

Preferred variants which form pores displaying an increased throughputcomprise mutations at the following positions:

-   -   D149, E185 and E203;    -   D149, E185, E201 and E203; or    -   D149, E185, D195, E201 and E203.

Preferred variants which form pores displaying an increased throughputcomprise:

-   -   D149N, E185N and E203N;    -   D149N, E185N, E201N and E203N;    -   D149N, E185R, D195N, E201N and E203N; or    -   D149N, E185R, D195N, E201R and E203N.

Preferred variants which form pores in which capture of thepolynucleotide is increased comprise the following mutations:

-   -   D43N/Y51T/F56Q;    -   E44N/Y51T/F56Q;    -   D43N/E44N/Y51T/F56Q;    -   Y51T/F56Q/Q62R;    -   D43N/Y51T/F56Q/Q62R;    -   E44N/Y51T/F56Q/Q62R; or    -   D43N/E44N/Y51T/F56Q/Q62R.

Preferred variants comprise the following mutations:

-   -   D149R/E185R/E201R/E203R or Y51T/F56Q/D149R/E185R/E201R/E203R;    -   D149N/E185N/E201N/E203N or Y51T/F56Q/D149N/E185N/E201N/E203N;    -   E201R/E203R or Y51T/F56Q/E201R/E203R    -   E201N/E203R or Y51T/F56Q/E201N/E203R;    -   E203R or Y51T/F56Q/E203R;    -   E203N or Y51T/F56Q/E203N;    -   E201R or Y51T/F56Q/E201R;    -   E201N or Y51T/F56Q/E201N;    -   E185R or Y51T/F56Q/E185R;    -   E185N or Y51T/F56Q/E185N;    -   D149R or Y51T/F56Q/D149R;    -   D149N or Y51T/F56Q/D149N;    -   R142E or Y51T/F56Q/R142E;    -   R142N or Y51T/F56Q/R142N;    -   R192E or Y51T/F56Q/R192E; or    -   R192N or Y51T/F56Q/R192N.

Preferred variants comprise the following mutations:

-   -   Y51A/F56Q/E101N/N102R;    -   Y51A/F56Q/R97N/N102G;    -   Y51A/F56Q/R97N/N102R;    -   Y51A/F56Q/R97N;    -   Y51A/F56Q/R97G;    -   Y51A/F56Q/R97L;    -   Y51A/F56Q/N102R;    -   Y51A/F56Q/N102F;    -   Y51A/F56Q/N102G;    -   Y51A/F56Q/E101R;    -   Y51A/F56Q/E101F;    -   Y51A/F56Q/E101N; or    -   Y51A/F56Q/E101G

The invention also provides a mutant CsgG monomer comprising a variantof the sequence shown in SEQ ID NO: 390, wherein the variant comprises amutation at T150. A preferred variant which forms a pore displaying anincreased insertion comprises T150I. A mutation at T150, such as T150I,may be combined with any of the mutations or combinations of mutationsdiscussed above.

The invention also provides a mutant CsgG monomer comprising a variantof the sequence shown in SEQ ID NO: 390 comprising the combination ofmutations present in a variant disclosed in the Examples.

Methods for introducing or substituting naturally-occurring amino acidsare well known in the art. For instance, methionine (M) may besubstituted with arginine (R) by replacing the codon for methionine(ATG) with a codon for arginine (CGT) at the relevant position in apolynucleotide encoding the mutant monomer. The polynucleotide can thenbe expressed as discussed below.

Methods for introducing or substituting non-naturally-occurring aminoacids are also well known in the art. For instance,non-naturally-occurring amino acids may be introduced by includingsynthetic aminoacyl-tRNAs in the IVTT system used to express the mutantmonomer. Alternatively, they may be introduced by expressing the mutantmonomer in E. coli that are auxotrophic for specific amino acids in thepresence of synthetic (i.e. non-naturally-occurring) analogues of thosespecific amino acids. They may also be produced by naked ligation if themutant monomer is produced using partial peptide synthesis.

Other Monomers of the Invention

In another embodiment, the invention provides a mutant CsgG monomercomprising a variant of the sequence shown in SEQ ID NO: 390, whereinthe variant comprises a mutation at one or more of positions Y51, N55and F56. The variant may comprise a mutation at Y51; N55; F56; Y51/N55;Y51/F56; N55/F56; or Y51/N55/F56. The variant preferably comprises amutation at Y51, N55 or F56. The variant may comprise any of thespecific mutations at one or more of positions Y51, N55 and F56discussed above and in any combination. One or more Y51, N55 and F56 maybe substituted with any amino acid. Y51 may be substituted with F, M, L,I, V, A, P, G, C, Q, N, T, S, E, D, K, H or R, such as A, S, T, N or Q.N55 may be substituted with F, M, L, I, V, A, P, G, C, Q, T, S, E, D, K,H or R, such as A, S, T or Q. F56 may be substituted with M, L, I, V, A,P, G, C, Q, N, T, S, E, D, K, H or R, such as A, S, T, N or Q.

The variant may further comprise one or more of the following: (i) oneor more mutations at the following positions (i.e. mutations at one ormore of the following positions) (i) N40, D43, E44, S54, S57, Q62, R97,E101, E124, E131, R142, T150 and R192; (iii) Q42R or Q42K; (iv) K49R;(v) N102R, N102F, N102Y or N102W; (vi) D149N, D149Q or D149R; (vii)E185N, E185Q or E185R; (viii) D195N, D195Q or D195R; (ix) E201N, E201Qor E201R; (x) E203N, E203Q or E203R; and (xi) deletion of one or more ofthe following positions F48, K49, P50, Y51, P52, A53, S54, N55, F56 andS57. The variant may comprise any of the combinations of (i) and (iii)to (xi) discussed above. The variant may comprise any of the embodimentsdiscussed above for (i) and (iii) to (xi).

Variants

In addition to the specific mutations discussed above, the variant mayinclude other mutations. Over the entire length of the amino acidsequence of SEQ ID NO: 390, a variant will preferably be at least 50%homologous to that sequence based on amino acid identity. Morepreferably, the variant may be at least 55%, at least 60%, at least 65%,at least 70%, at least 75%, at least 80%, at least 85%, at least 90% andmore preferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 390 over the entiresequence. There may be at least 80%, for example at least 85%, 90% or95%, amino acid identity over a stretch of 100 or more, for example 125,150, 175 or 200 or more, contiguous amino acids (“hard homology”).

Standard methods in the art may be used to determine homology. Forexample the UWGCG Package provides the BESTFIT program which can be usedto calculate homology, for example used on its default settings(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent residues or correspondingsequences (typically on their default settings)), for example asdescribed in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. Fet al (1990) J Mol Biol 215:403-10. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 390 is the wild-type CsgG monomer from Escherichia coli Str.K-12 substr. MC4100. The variant of SEQ ID NO: 390 may comprise any ofthe substitutions present in another CsgG homologue. Preferred CsgGhomologues are shown in SEQ ID NOs: 391 to 395 and 414 to 429. Thevariant may comprise combinations of one or more of the substitutionspresent in SEQ ID NOs: 391 to 395 and 414 to 429 compared with SEQ IDNO: 390.

Amino acid substitutions may be made to the amino acid sequence of SEQID NO: 390 in addition to those discussed above, for example up to 1, 2,3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replaceamino acids with other amino acids of similar chemical structure,similar chemical properties or similar side-chain volume. The aminoacids introduced may have similar polarity, hydrophilicity,hydrophobicity, basicity, acidity, neutrality or charge to the aminoacids they replace. Alternatively, the conservative substitution mayintroduce another amino acid that is aromatic or aliphatic in the placeof a pre-existing aromatic or aliphatic amino acid. Conservative aminoacid changes are well-known in the art and may be selected in accordancewith the properties of the 20 main amino acids as defined in Table 1below. Where amino acids have similar polarity, this can also bedetermined by reference to the hydropathy scale for amino acid sidechains in Table 2.

TABLE 1 Chemical properties of amino acids Ala aliphatic, hydrophobic,neutral Met hydrophobic, neutral Cys polar, hydrophobic, neutral Asnpolar, hydrophilic, neutral Asp polar, hydrophilic, charged (−) Prohydrophobic, neutral Glu polar, hydrophilic, charged (−) Gln polar,hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar,hydrophilic, charged (+) Gly aliphatic, neutral Ser polar, hydrophilic,neutral His aromatic, polar, hydrophilic, Thr polar, hydrophilic,neutral charged (+) Ile aliphatic, hydrophobic, neutral Val aliphatic,hydrophobic, neutral Lys polar, hydrophilic, charged(+) Trp aromatic,hydrophobic, neutral Leu aliphatic, hydrophobic, neutral Tyr aromatic,polar, hydrophobic

TABLE 2 Hydropathy scale Side Chain Hydropathy Ile 4.5 Val 4.2 Leu 3.8Phe 2.8 Cys 2.5 Met 1.9 Ala 1.8 Gly −0.4 Thr −0.7 Ser −0.8 Trp −0.9 Tyr−1.3 Pro −1.6 His −3.2 Glu −3.5 Gln −3.5 Asp −3.5 Asn −3.5 Lys −3.9 Arg−4.5

One or more amino acid residues of the amino acid sequence of SEQ ID NO:390 may additionally be deleted from the polypeptides described above.Up to 1, 2, 3, 4, 5, 10, 20 or 30 or more residues may be deleted.

Variants may include fragments of SEQ ID NO: 390. Such fragments retainpore forming activity. Fragments may be at least 50, at least 100, atleast 150, at least 200 or at least 250 amino acids in length. Suchfragments may be used to produce the pores. A fragment preferablycomprises the membrane spanning domain of SEQ ID NO: 390, namelyK135-Q153 and S183-S208.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theamino terminal or carboxy terminal of the amino acid sequence of SEQ IDNO: 390 or polypeptide variant or fragment thereof. The extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer, for example up to 50 or 100amino acids. A carrier protein may be fused to an amino acid sequenceaccording to the invention. Other fusion proteins are discussed in moredetail below.

As discussed above, a variant is a polypeptide that has an amino acidsequence which varies from that of SEQ ID NO: 390 and which retains itsability to form a pore. A variant typically contains the regions of SEQID NO: 390 that are responsible for pore formation. The pore formingability of CsgG, which contains a β-barrel, is provided by β-sheets ineach subunit. A variant of SEQ ID NO: 390 typically comprises theregions in SEQ ID NO: 390 that form β-sheets, namely K135-Q153 andS183-S208. One or more modifications can be made to the regions of SEQID NO: 390 that form β-sheets as long as the resulting variant retainsits ability to form a pore. A variant of SEQ ID NO: 390 preferablyincludes one or more modifications, such as substitutions, additions ordeletions, within its α-helices and/or loop regions.

The monomers derived from CsgG may be modified to assist theiridentification or purification, for example by the addition of astreptavidin tag or by the addition of a signal sequence to promotetheir secretion from a cell where the monomer does not naturally containsuch a sequence. Other suitable tags are discussed in more detail below.The monomer may be labelled with a revealing label. The revealing labelmay be any suitable label which allows the monomer to be detected.Suitable labels are described below.

The monomer derived from CsgG may also be produced using D-amino acids.For instance, the monomer derived from CsgG may comprise a mixture ofL-amino acids and D-amino acids. This is conventional in the art forproducing such proteins or peptides.

The monomer derived from CsgG contains one or more specificmodifications to facilitate nucleotide discrimination. The monomerderived from CsgG may also contain other non-specific modifications aslong as they do not interfere with pore formation. A number ofnon-specific side chain modifications are known in the art and may bemade to the side chains of the monomer derived from CsgG. Suchmodifications include, for example, reductive alkylation of amino acidsby reaction with an aldehyde followed by reduction with NaBH₄,amidination with methylacetimidate or acylation with acetic anhydride.

The monomer derived from CsgG can be produced using standard methodsknown in the art. The monomer derived from CsgG may be madesynthetically or by recombinant means. For example, the monomer may besynthesised by in vitro translation and transcription (IVTT). Suitablemethods for producing pores and monomers are discussed in InternationalApplication Nos. PCT/GB09/001690 (published as WO 2010/004273),PCT/GB09/001679 (published as WO 2010/004265) or PCT/GB10/000133(published as WO 2010/086603). Methods for inserting pores intomembranes are discussed.

In some embodiments, the mutant monomer is chemically modified. Themutant monomer can be chemically modified in any way and at any site.The mutant monomer is preferably chemically modified by attachment of amolecule to one or more cysteines (cysteine linkage), attachment of amolecule to one or more lysines, attachment of a molecule to one or morenon-natural amino acids, enzyme modification of an epitope ormodification of a terminus. Suitable methods for carrying out suchmodifications are well-known in the art. The mutant monomer may bechemically modified by the attachment of any molecule. For instance, themutant monomer may be chemically modified by attachment of a dye or afluorophore.

In some embodiments, the mutant monomer is chemically modified with amolecular adaptor that facilitates the interaction between a porecomprising the monomer and a target nucleotide or target polynucleotidesequence. The presence of the adaptor improves the host-guest chemistryof the pore and the nucleotide or polynucleotide sequence and therebyimproves the sequencing ability of pores formed from the mutant monomer.The principles of host-guest chemistry are wall-known in the art. Theadaptor has an effect on the physical or chemical properties of the porethat improves its interaction with the nucleotide or polynucleotidesequence. The adaptor may alter the charge of the barrel or channel ofthe pore or specifically interact with or bind to the nucleotide orpolynucleotide sequence thereby facilitating its interaction with thepore.

The molecular adaptor is preferably a cyclic molecule, a cyclodextrin, aspecies that is capable of hybridization, a DNA binder or intercheletor,a peptide or peptide analogue, a synthetic polymer, an aromatic planarmolecule, a small positively-charged molecule or a small moleculecapable of hydrogen-bonding.

The adaptor may be cyclic. A cyclic adaptor preferably has the samesymmetry as the pore. The adaptor preferably has eight-fold or nine-foldsymmetry since CsgG typically has eight or nine subunits around acentral axis. This is discussed in more detail below.

The adaptor typically interacts with the nucleotide or polynucleotidesequence via host-guest chemistry. The adaptor is typically capable ofinteracting with the nucleotide or polynucleotide sequence. The adaptorcomprises one or more chemical groups that are capable of interactingwith the nucleotide or polynucleotide sequence. The one or more chemicalgroups preferably interact with the nucleotide or polynucleotidesequence by non-covalent interactions, such as hydrophobic interactions,hydrogen bonding, Van der Waal's forces, π-cation interactions and/orelectrostatic forces. The one or more chemical groups that are capableof interacting with the nucleotide or polynucleotide sequence arepreferably positively charged. The one or more chemical groups that arecapable of interacting with the nucleotide or polynucleotide sequencemore preferably comprise amino groups. The amino groups can be attachedto primary, secondary or tertiary carbon atoms. The adaptor even morepreferably comprises a ring of amino groups, such as a ring of 6, 7 or 8amino groups. The adaptor most preferably comprises a ring of eightamino groups. A ring of protonated amino groups may interact withnegatively charged phosphate groups in the nucleotide or polynucleotidesequence.

The correct positioning of the adaptor within the pore can befacilitated by host-guest chemistry between the adaptor and the porecomprising the mutant monomer. The adaptor preferably comprises one ormore chemical groups that are capable of interacting with one or moreamino acids in the pore. The adaptor more preferably comprises one ormore chemical groups that are capable of interacting with one or moreamino acids in the pore via non-covalent interactions, such ashydrophobic interactions, hydrogen bonding, Van der Waal's forces,π-cation interactions and/or electrostatic forces. The chemical groupsthat are capable of interacting with one or more amino acids in the poreare typically hydroxyls or amines. The hydroxyl groups can be attachedto primary, secondary or tertiary carbon atoms. The hydroxyl groups mayform hydrogen bonds with uncharged amino acids in the pore. Any adaptorthat facilitates the interaction between the pore and the nucleotide orpolynucleotide sequence can be used.

Suitable adaptors include, but are not limited to, cyclodextrins, cyclicpeptides and cucurbiturils. The adaptor is preferably a cyclodextrin ora derivative thereof. The cyclodextrin or derivative thereof may be anyof those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am.Chem. Soc. 116, 6081-6088. The adaptor is more preferablyheptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-βCD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD). The guanidinogroup in gu₇-βCD has a much higher pKa than the primary amines inam₇-βCD and so it is more positively charged. This gu₇-βCD adaptor maybe used to increase the dwell time of the nucleotide in the pore, toincrease the accuracy of the residual current measured, as well as toincrease the base detection rate at high temperatures or low dataacquisition rates.

If a succinimidyl 3-(2-pyridyldithio)propionate (SPDP) crosslinker isused as discussed in more detail below, the adaptor is preferablyheptakis(6-deoxy-6-amino)-N-mono(2-pyridyl)dithiopropanoyl-β-cyclodextrin(am₆amPDP₁-βCD).

More suitable adaptors include γ-cyclodextrins, which comprise 9 sugarunits (and therefore have nine-fold symmetry). The γ-cyclodextrin maycontain a linker molecule or may be modified to comprise all or more ofthe modified sugar units used in the β-cyclodextrin examples discussedabove.

The molecular adaptor is preferably covalently attached to the mutantmonomer. The adaptor can be covalently attached to the pore using anymethod known in the art. The adaptor is typically attached via chemicallinkage. If the molecular adaptor is attached via cysteine linkage, theone or more cysteines have preferably been introduced to the mutant, forinstance in the barrel, by substitution. The mutant monomer may bechemically modified by attachment of a molecular adaptor to one or morecysteines in the mutant monomer. The one or more cysteines may benaturally-occurring, i.e. at positions 1 and/or 215 in SEQ ID NO: 390.Alternatively, the mutant monomer may be chemically modified byattachment of a molecule to one or more cysteines introduced at otherpositions. The cysteine at position 215 may be removed, for instance bysubstitution, to ensure that the molecular adaptor does not attach tothat position rather than the cysteine at position 1 or a cysteineintroduced at another position.

The reactivity of cysteine residues may be enhanced by modification ofthe adjacent residues. For instance, the basic groups of flankingarginine, histidine or lysine residues will change the pKa of thecysteines thiol group to that of the more reactive S⁻ group. Thereactivity of cysteine residues may be protected by thiol protectivegroups such as dTNB. These may be reacted with one or more cysteineresidues of the mutant monomer before a linker is attached.

The molecule may be attached directly to the mutant monomer. Themolecule is preferably attached to the mutant monomer using a linker,such as a chemical crosslinker or a peptide linker.

Suitable chemical crosslinkers are well-known in the art. Preferredcrosslinkers include 2,5-dioxopyrrolidin-1-yl3-(pyridin-2-yldisulfanyl)propenoate, 2,5-dioxopyrrolidin-1-yl4-(pyridin-2-yldisulfanyl)butanoate and 2,5-dioxopyrrolidin-1-yl8-(pyridin-2-yldisulfanyl)octananoate. The most preferred crosslinker issuccinimidyl 3-(2-pyridyldithio)propionate (SPDP). Typically, themolecule is covalently attached to the bifunctional crosslinker beforethe molecule/crosslinker complex is covalently attached to the mutantmonomer but it is also possible to covalently attach the bifunctionalcrosslinker to the monomer before the bifunctional crosslinker/monomercomplex is attached to the molecule.

The linker is preferably resistant to dithiothreitol (DTT). Suitablelinkers include, but are not limited to, iodoacetamide-based andMaleimide-based linkers.

In other embodiment, the monomer may be attached to a polynucleotidebinding protein. This forms a modular sequencing system that may be usedin the methods of sequencing of the invention. Polynucleotide bindingproteins are discussed below.

The polynucleotide binding protein is preferably covalently attached tothe mutant monomer. The protein can be covalently attached to themonomer using any method known in the art. The monomer and protein maybe chemically fused or genetically fused. The monomer and protein aregenetically fused if the whole construct is expressed from a singlepolynucleotide sequence. Genetic fusion of a monomer to a polynucleotidebinding protein is discussed in International Application No.PCT/GB09/001679 (published as WO 2010/004265).

If the polynucleotide binding protein is attached via cysteine linkage,the one or more cysteines have preferably been introduced to the mutantby substitution. The one or more cysteines are preferably introducedinto loop regions which have low conservation amongst homologuesindicating that mutations or insertions may be tolerated. They aretherefore suitable for attaching a polynucleotide binding protein. Insuch embodiments, the naturally-occurring cysteine at position 251 maybe removed. The reactivity of cysteine residues may be enhanced bymodification as described above.

The polynucleotide binding protein may be attached directly to themutant monomer or via one or more linkers. The molecule may be attachedto the mutant monomer using the hybridization linkers described inInternational Application No. PCT/GB10/000132 (published as WO2010/086602). Alternatively, peptide linkers may be used. Peptidelinkers are amino acid sequences. The length, flexibility andhydrophilicity of the peptide linker are typically designed such that itdoes not to disturb the functions of the monomer and molecule. Preferredflexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10or 16, serine and/or glycine amino acids. More preferred flexiblelinkers include (SG)₁, (SG)₂, (SG)₃, (SG)₄, (SG)₅ and (SG)₈ wherein S isserine and G is glycine. Preferred rigid linkers are stretches of 2 to30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigidlinkers include (P)₁₂ wherein P is proline.

The mutant monomer may be chemically modified with a molecular adaptorand a polynucleotide binding protein.

The molecule (with which the monomer is chemically modified) may beattached directly to the monomer or attached via a linker as disclosedin International Application Nos. PCT/GB09/001690 (published as WO2010/004273), PCT/GB09/001679 (published as WO 20101004265) orPCT/GB10/000133 (published as WO 2010/086603).

Any of the proteins described herein, such as the mutant monomers andpores of the invention, may be modified to assist their identificationor purification, for example by the addition of histidine residues (ahis tag), aspartic acid residues (an asp tag), a streptavidin tag, aflag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of asignal sequence to promote their secretion from a cell where thepolypeptide does not naturally contain such a sequence. An alternativeto introducing a genetic tag is to chemically react a tag onto a nativeor engineered position on the protein. An example of this would be toreact a gel-shift reagent to a cysteine engineered on the outside of theprotein. This has been demonstrated as a method for separating hemolysinhetero-oligomers (Chem Biol. 1997 July; 4(7):497-505).

Any of the proteins described herein, such as the mutant monomers andpores of the invention, may be labeled with a revealing label. Therevealing label may be any suitable label which allows the protein to bedetected. Suitable labels include, but are not limited to, fluorescentmolecules, radioisotopes, e.g. ¹²⁵I, ³⁵S, enzymes, antibodies, antigens,polynucleotides and ligands such as biotin.

Any of the proteins described herein, such as the monomers or pores ofthe invention, may be made synthetically or by recombinant means. Forexample, the protein may be synthesised by in vitro translation andtranscription (IVTT). The amino acid sequence of the protein may bemodified to include non-naturally occurring amino acids or to increasethe stability of the protein. When a protein is produced by syntheticmeans, such amino acids may be introduced during production. The proteinmay also be altered following either synthetic or recombinantproduction.

Proteins may also be produced using D-amino acids. For instance, theprotein may comprise a mixture of L-amino acids and D-amino acids. Thisis conventional in the art for producing such proteins or peptides.

The protein may also contain other non-specific modifications as long asthey do not interfere with the function of the protein. A number ofnon-specific side chain modifications are known in the art and may bemade to the side chains of the protein(s). Such modifications include,for example, reductive alkylation of amino acids by reaction with analdehyde followed by reduction with NaBH₄, amidination withmethylacetimidate or acylation with acetic anhydride.

Any of the proteins described herein, including the monomers and poresof the invention, can be produced using standard methods known in theart. Polynucleotide sequences encoding a protein may be derived andreplicated using standard methods in the art. Polynucleotide sequencesencoding a protein may be expressed in a bacterial host cell usingstandard techniques in the art. The protein may be produced in a cell byin situ expression of the polypeptide from a recombinant expressionvector. The expression vector optionally carries an inducible promoterto control the expression of the polypeptide. These methods aredescribed in Sambrook, J. and Russell, D. (2001). Molecular Cloning: ALaboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.

Proteins may be produced in large scale following purification by anyprotein liquid chromatography system from protein producing organisms orafter recombinant expression. Typical protein liquid chromatographysystems include FPLC, AKTA systems, the Bio-Cad system, the Bio-RadBioLogic system and the Gilson HPLC system.

Constructs

The invention also provides a construct comprising two or morecovalently attached CsgG monomers, wherein at least one of the monomersis a mutant monomer of the invention. The construct of the inventionretains its ability to form a pore. This may be determined as discussedabove. One or more constructs of the invention may be used to form poresfor characterising, such as sequencing, polynucleotides. The constructmay comprise at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9 or at least 10 monomers. Theconstruct preferably comprises two monomers. The two or more monomersmay be the same or different.

At least one monomer in the construct is a mutant monomer of theinvention. 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 ormore, 8 or more, 9 or more or 10 or more monomers in the construct maybe mutant monomers of the invention. All of the monomers in theconstruct are preferably mutant monomers of the invention. The mutantmonomers may be the same or different. In a preferred embodiment, theconstruct comprises two mutant monomers of the invention.

The mutant monomers of the invention in the construct are preferablyapproximately the same length or are the same length. The barrels of themutant monomers of the invention in the construct are preferablyapproximately the same length or are the same length. Length may bemeasured in number of amino acids and/or units of length.

The construct may comprise one or more monomers which are not mutantmonomers of the invention. CsgG mutant monomers which are non mutantmonomers of the invention include monomers comprising SEQ ID NO: 390,391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428 or 429 or a comparative variant of SEQ IDNO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420,421, 422, 423, 424, 425, 426, 427, 428 or 429 in which none of the aminoacids/positions discussed above have been been mutated. At least onemonomer in the construct may comprise SEQ ID NO: 390, 391, 392, 393,394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428 or 429 or a comparative variant of the sequence shown inSEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419,420, 421, 422, 423, 424, 425, 426, 427, 428 or 429. A comparativevariant of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417,418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 is at least50% homologous to 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418,419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429, 40 or 41 overits entire sequence based on amino acid identity. More preferably, thecomparative variant may be at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90% andmore preferably at least 95%, 97% or 99% homologous based on amino acididentity to the amino acid sequence of SEQ ID NO: 390, 391, 392, 393,394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428 or 429 over the entire sequence.

The monomers in the construct are preferably genetically fused. Monomersare genetically fused if the whole construct is expressed from a singlepolynucleotide sequence. The coding sequences of the monomers may becombined in any way to form a single polynucleotide sequence encodingthe construct.

The monomers may be genetically fused in any configuration. The monomersmay be fused via their terminal amino acids. For instance, the aminoterminus of the one monomer may be fused to the carboxy terminus ofanother monomer. The second and subsequent monomers in the construct (inthe amino to carboxy direction) may comprise a methionine at their aminoterminal ends (each of which is fused to the carboxy terminus of theprevious monomer). For instance, if M is a monomer (without an aminoterminal methionine) and mM is a monomer with an amino terminalmethionine, the construct may comprise the sequence M-mM, M-mM-mM orM-mM-mM-mM. The presences of these methionines typically results fromthe expression of the start codons (i.e. ATGs) at the 5′ end of thepolynucleotides encoding the second or subsequent monomers within thepolynucleotide encoding entire construct. The first monomer in theconstruct (in the amino to carboxy direction) may also comprise amethionine (e.g. mM-mM, mM-mM-mM or mM-mM-mM-mM).

The two or more monomers may be genetically fused directly together. Themonomers are preferably genetically fused using a linker. The linker maybe designed to constrain the mobility of the monomers. Preferred linkersare amino acid sequences (i.e. peptide linkers). Any of the peptidelinkers discussed above may be used.

In another preferred embodiment, the monomers are chemically fused. Twomonomers are chemically fused if the two parts are chemically attached,for instance via a chemical crosslinker. Any of the chemicalcrosslinkers discussed above may be used. The linker may be attached toone or more cysteine residues introduced into a mutant monomer of theinvention. Alternatively, the linker may be attached to a terminus ofone of the monomers in the construct.

If a construct contains different monomers, crosslinkage of monomers tothemselves may be prevented by keeping the concentration of linker in avast excess of the monomers. Alternatively, a “lock and key” arrangementmay be used in which two linkers are used. Only one end of each linkermay react together to form a longer linker and the other ends of thelinker each react with a different monomers. Such linkers are describedin International Application No. PCT/GB10/000132 (published as WO2010/086602).

Polynucleotides

The present invention also provides polynucleotide sequences whichencode a mutant monomer of the invention. The mutant monomer may be anyof those discussed above. The polynucleotide sequence preferablycomprises a sequence at least 50%, 60%, 70%, 80%, 90% or 95% homologousbased on nucleotide identity to the sequence of SEQ ID NO: 389 over theentire sequence. There may be at least 80%, for example at least 85%,90% or 95% nucleotide identity over a stretch of 300 or more, forexample 375, 450, 525 or 600 or more, contiguous nucleotides (“hardhomology”). Homology may be calculated as described above. Thepolynucleotide sequence may comprise a sequence that differs from SEQ IDNO: 389 on the basis of the degeneracy of the genetic code.

The present invention also provides polynucleotide sequences whichencode any of the genetically fused constructs of the invention. Thepolynucleotide preferably comprises two or more variants of the sequenceshown in SEQ ID NO: 389. The polynucleotide sequence preferablycomprises two or more sequences having at least 50%, 60%, 70%, 80%, 90%or 95% homology to SEQ ID NO: 389 based on nucleotide identity over theentire sequence. There may be at least 80%, for example at least 85%,90% or 95% nucleotide identity over a stretch of 600 or more, forexample 750, 900, 1050 or 1200 or more, contiguous nucleotides (“hardhomology”). Homology may be calculated as described above.

Polynucleotide sequences may be derived and replicated using standardmethods in the art. Chromosomal DNA encoding wild-type CsgG may beextracted from a pore producing organism, such as Escherichia coli. Thegene encoding the pore subunit may be amplified using PCR involvingspecific primers. The amplified sequence may then undergo site-directedmutagenesis. Suitable methods of site-directed mutagenesis are known inthe art and include, for example, combine chain reaction.Polynucleotides encoding a construct of the invention can be made usingwell-known techniques, such as those described in Sambrook, J. andRussell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition.Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The resulting polynucleotide sequence may then be incorporated into arecombinant replicable vector such as a cloning vector. The vector maybe used to replicate the polynucleotide in a compatible host cell. Thuspolynucleotide sequences may be made by introducing a polynucleotideinto a replicable vector, introducing the vector into a compatible hostcell, and growing the host cell under conditions which bring aboutreplication of the vector. The vector may be recovered from the hostcell. Suitable host cells for cloning of polynucleotides are known inthe art and described in more detail below.

The polynucleotide sequence may be cloned into suitable expressionvector. In an expression vector, the polynucleotide sequence istypically operably linked to a control sequence which is capable ofproviding for the expression of the coding sequence by the host cell.Such expression vectors can be used to express a pore subunit.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. Multiple copies of the same or different polynucleotidesequences may be introduced into the vector.

The expression vector may then be introduced into a suitable host cell.Thus, a mutant monomer or construct of the invention can be produced byinserting a polynucleotide sequence into an expression vector,introducing the vector into a compatible bacterial host cell, andgrowing the host cell under conditions which bring about expression ofthe polynucleotide sequence. The recombinantly-expressed monomer orconstruct may self-assemble into a pore in the host cell membrane.Alternatively, the recombinant pore produced in this manner may beremoved from the host cell and inserted into another membrane. Whenproducing pores comprising at least two different monomers orconstructs, the different monomers or constructs may be expressedseparately in different host cells as described above, removed from thehost cells and assembled into a pore in a separate membrane, such as arabbit cell membrane or a synthetic membrane.

The vectors may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide sequence and optionally a regulator of thepromoter. The vectors may contain one or more selectable marker genes,for example a tetracycline resistance gene. Promoters and otherexpression regulation signals may be selected to be compatible with thehost cell for which the expression vector is designed. A T7, trc, lac,are or λ_(L) promoter is typically used.

The host cell typically expresses the monomer or construct at a highlevel. Host cells transformed with a polynucleotide sequence will bechosen to be compatible with the expression vector used to transform thecell. The host cell is typically bacterial and preferably Escherichiacoli. Any cell with a λ DE3 lysogen, for example C41 (DE3), BL21 (DE3),JM109 (DE3), B834 (DE3), TUNER, Origami and Origami B, can express avector comprising the T7 promoter. In addition to the conditions listedabove any of the methods cited in Cao et al, 2014, PNAS, Structure ofthe nonameric bacterial amyloid secretion channel, doi-1411942111 andGoyal et al, 2014, Nature, 516, 250-253 structural and mechanisticinsights into the bacterial amyloid secretion channel CsgG may be usedto express the CsgG proteins.

The invention also comprises a method of producing a mutant monomer ofthe invention or a construct of the invention. The method comprisesexpressing a polynucleotide of the invention in a suitable host cell.The polynucleotide is preferably part of a vector and is preferablyoperably linked to a promoter.

Pores

The invention also provides various pores. The pores of the inventionare ideal for characterising, such as sequencing, polynucleotidesequences because they can discriminate between different nucleotideswith a high degree of sensitivity. The pores can surprisinglydistinguish between the four nucleotides in DNA and RNA. The pores ofthe invention can even distinguish between methylated and unmethylatednucleotides. The base resolution of pores of the invention issurprisingly high. The pores show almost complete separation of all fourDNA nucleotides. The pores further discriminate between deoxycytidinemonophosphate (dCMP) and methyl-dCMP based on the dwell time in the poreand the current flowing through the pore.

The pores of the invention can also discriminate between differentnucleotides under a range of conditions. In particular, the pores willdiscriminate between nucleotides under conditions that are favourable tothe characterising, such as sequencing, of nucleic acids. The extent towhich the pores of the invention can discriminate between differentnucleotides can be controlled by altering the applied potential, thesalt concentration, the buffer, the temperature and the presence ofadditives, such as urea, betaine and DTT. This allows the function ofthe pores to be fine-tuned, particularly when sequencing. This isdiscussed in more detail below. The pores of the invention may also beused to identify polynucleotide polymers from the interaction with oneor more monomers rather than on a nucleotide by nucleotide basis.

A pore of the invention may be isolated, substantially isolated,purified or substantially purified. A pore of the invention is isolatedor purified if it is completely free of any other components, such aslipids or other pores. A pore is substantially isolated if it is mixedwith carriers or diluents which will not interfere with its intendeduse. For instance, a pore is substantially isolated or substantiallypurified if it is present in a form that comprises less than 10%, lessthan 5%, less than 2% or less than 1% of other components, such astriblock copolymers, lipids or other pores. Alternatively, a pore of theinvention may be present in a membrane. Suitable membranes are discussedbelow.

A pore of the invention may be present as an individual or single pore.Alternatively, a pore of the invention may be present in a homologous orheterologous population of two or more pores.

Homo-Oligomeric Pores

The invention also provides a homo-oligomeric pore derived from CsgGcomprising identical mutant monomers of the invention. Thehomo-oligomeric pore may comprise any of the mutants of the invention.The homo-oligomeric pore of the invention is ideal for characterising,such as sequencing, polynucleotides. The homo-oligomeric pore of theinvention may have any of the advantages discussed above.

The homo-oligomeric pore may contain any number of mutant monomers. Thepore typically comprises at least 7, at least 8, at least 9 or at least10 identical mutant monomers, such as 7, 8, 9 or 10 mutant monomers. Thepore preferably comprises eight or nine identical mutant monomers. Oneor more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the mutant monomers ispreferably chemically modified as discussed above.

Methods for making pores are discussed in more detail below.

Hetero-Oligomeric Pores

The invention also provides a hetero-oligomeric pore derived from CsgGcomprising at least one mutant monomer of the invention. Thehetero-oligomeric pore of the invention is ideal for characterising,such as sequencing, polynucleotides. Hetero-oligomeric pores can be madeusing methods known in the art (e.g. Protein Sci. 2002 July;11(7):1813-24).

The hetero-oligomeric pore contains sufficient monomers to form thepore. The monomers may be of any type. The pore typically comprises atleast 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9or 10 monomers. The pore preferably comprises eight or nine monomers.

In a preferred embodiment, all of the monomers (such as 10, 9, 8 or 7 ofthe monomers) are mutant monomers of the invention and at least one ofthem differs from the others. In a more preferred embodiment, the porecomprises eight or nine mutant monomers of the invention and at leastone of them differs from the others. They may all differ from oneanother.

The mutant monomers of the invention in the pore are preferablyapproximately the same length or are the same length. The barrels of themutant monomers of the invention in the pore are preferablyapproximately the same length or are the same length. Length may bemeasured in number of amino acids and/or units of length.

In another preferred embodiment, at least one of the mutant monomers isnot a mutant monomer of the invention. In this embodiment, the remainingmonomers are preferably mutant monomers of the invention. Hence, thepore may comprise 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutant monomers of theinvention. Any number of the monomers in the pore may not be a mutantmonomer of the invention. The pore preferably comprises seven or eightmutant monomers of the invention and a monomer which is not a monomer ofthe invention. The mutant monomers of the invention may be the same ordifferent.

The mutant monomers of the invention in the construct are preferablyapproximately the same length or are the same length. The barrels of themutant monomers of the invention in the construct are preferablyapproximately the same length or are the same length. Length may bemeasured in number of amino acids and/or units of length.

The pore may comprise one or more monomers which are not mutant monomersof the invention. CsgG monomers which are not mutant monomers of theinvention include monomers comprising SEQ ID NO: 390, 391, 392, 393,394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425,426, 427, 428 or 429 or a comparative variant of SEQ ID NO: 390, 391,392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428 or 429 in which none of the aminoacids/positions discussed above in relation to the invention have beenmutated/substituted. A comparative variant of SEQ ID NO: 390, 391, 392,393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,425, 426, 427, 428 or 429 is typically at least 50% homologous to SEQ IDNO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420,421, 422, 423, 424, 425, 426, 427, 428 or 429 over its entire sequencebased on amino acid identity. More preferably, the comparative variantmay be at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% and more preferably atleast 95%, 97% or 99% homologous based on amino acid identity to theamino acid sequence of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or429 over the entire sequence.

In all the embodiments discussed above, one or more, such as 2, 3, 4, 5,6, 7, 8, 9 or 10, of the mutant monomers is preferably chemicallymodified as discussed above.

Methods for making pores are discussed in more detail below.

Construct-Containing Pores

The invention also provides a pore comprising at least one construct ofthe invention. A construct of the invention comprises two or morecovalently attached monomers derived from CsgG wherein at least one ofthe monomers is a mutant monomer of the invention. In other words, aconstruct must contain more than one monomer. The pore containssufficient constructs and, if necessary, monomers to form the pore. Forinstance, an octameric pore may comprise (a) four constructs eachcomprising two constructs, (b) two constructs each comprising fourmonomers or (b) one construct comprising two monomers and six monomersthat do not form part of a construct. For instance, an nonameric poremay comprise (a) four constructs each comprising two constructs and onemonomer that does not form part of a construct, (b) two constructs eachcomprising four monomers and a monomer that does not form part of aconstruct or (b) one construct comprising two monomers and sevenmonomers that do not form part of a construct. Other combinations ofconstructs and monomers can be envisaged by the skilled person.

At least two of the monomers in the pore are in the form of a constructof the invention. The construct, and hence the pore, comprises at leastone mutant monomer of the invention. The pore typically comprises atleast 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9or 10 monomers, in total (at least two of which must be in a construct).The pore preferably comprises eight or nine monomers (at least two ofwhich must be in a construct).

The construct containing pore may be a homo-oligomer (i.e. includeidentical constructs) or be a hetero-oligomer (i.e. where at least oneconstruct differs from the others).

A pore typically contains (a) one construct comprising two monomers and(b) 5, 6, 7 or 8 monomers. The construct may be any of those discussedabove. The monomers may be any of those discussed above, includingmutant monomers of the invention, monomers comprising SEQ ID NO: 390,391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422,423, 424, 425, 426, 427, 428 or 429 and mutant monomers comprising acomparative variant of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or429 as discussed above.

Another typical pore comprises more than one construct of the invention,such as two, three or four constructs of the invention. If necessary,such pores further comprise sufficient additional monomers or constructsto form the pore. The additional monomer(s) may be any of thosediscussed above, including mutant monomers of the invention, monomerscomprising SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417,418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 and mutantmonomers comprising a comparative variant of SEQ ID NO: 390, 391, 392,393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,425, 426, 427, 428 or 429 as discussed above. The additionalconstruct(s) may be any of those discussed above or may be a constructcomprising two or more covalently attached CsgG monomers each comprisinga monomer comprising SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415,416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429or a comparative variant of SEQ ID NO: 390, 391, 392, 393, 394, 395,414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,428 or 429 as discussed above.

A further pore of the invention comprises only constructs comprising 2monomers, for example a pore may comprise 4, 5, 6, 7 or 8 constructscomprising 2 monomers. At least one construct is a construct of theinvention, i.e. at least one monomer in the at least one construct, andpreferably each monomer in the at least one construct, is a mutantmonomer of the invention. All of the constructs comprising 2 monomersmay be constructs of the invention.

A specific pore according to the invention comprises four constructs ofthe invention each comprising two monomers, wherein at least one monomerin each construct, and preferably each monomer in each construct, is amutant monomer of the invention. The constructs may oligomerise into apore with a structure such that only one monomer of each constructcontributes to the channel of the pore. Typically the other monomers ofthe construct will be on the outside of the channel of the pore. Forexample, pores of the invention may comprise 7, 8, 9 or 10 constructscomprising 2 monomers where the channel comprises 7, 8, 9 or 10monomers.

Mutations can be introduced into the construct as described above. Themutations may be alternating, i.e. the mutations are different for eachmonomer within a two monomer construct and the constructs are assembledas a homo-oligomer resulting in alternating modifications. In otherwords, monomers comprising MutA and MutB are fused and assembled to forman A-B:A-B:A-B:A-B pore. Alternatively, the mutations may beneighbouring, i.e. identical mutations are introduced into two monomersin a construct and this is then oligomerised with different mutantmonomers or constructs. In other words, monomers comprising MutA arefused follow by oligomerisation with MutB-containing monomers to formA-A:B:B:B:B:B:B.

One or more of the monomers of the invention in a construct-containingpore may be chemically-modified as discussed above.

Analyte Characterisation

The invention provides a method of determining the presence, absence orone or more characteristics of a target analyte. The method involvescontacting the target analyte with a CsgG pore or a mutant thereof, suchas a pore of the invention, such that the target analyte moves withrespect to, such as through, the pore and taking one or moremeasurements as the analyte moves with respect to the pore and therebydetermining the presence, absence or one or more characteristics of theanalyte. The target analyte may also be called the template analyte orthe analyte of interest.

The method comprises contacting the target analyte with a CsgG pore or amutant thereof, such as a pore of the invention, such that the targetanalyte moves through the pore. The pore typically comprises at least 7,at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10monomers. The pore preferably comprises eight or nine identicalmonomers. One or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of themonomers is preferably chemically modified as discussed above.

The CsgG pore may be derived from any organism. The CsgG pore maycomprise monomers comprising the sequence shown in SEQ ID NO: 390, 391,392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428 or 429. The CsgG pore may comprise at least 7,at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10monomers, each comprising the sequence shown in SEQ ID NO: 390, 391,392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428 or 429 (i.e. the pore is a homo-oligomercomprising identical monomers from the same organism). The CsgG maycomprise any combination of monomers each comprising a sequence shown inSEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419,420, 421, 422, 423, 424, 425, 426, 427, 428 or 429. For instance, thepore may comprise 7 monomers comprising the sequence shown in SEQ ID NO:390 and two monomers comprising the sequence shown in SEQ ID NO: 391.

The CsgG mutant may comprise any number of mutant monomers, such as atleast 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9or 10 monomers. The mutant monomers may comprise a comparative variantof the sequence shown in SEQ ID NO: 390, 391, 392, 393, 394, 395, 414,415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or429. Comparative variants are discussed above. Comparative variants mustbe capable of forming a pore and may have any of the % homologiesdiscussed above with reference to the pores of the invention.

The CsgG mutant preferably comprises nine monomers and at least one ofthe monomers is a variant of the sequence shown in SEQ ID NO: 390comprising (a) mutations at one or more of the following positions N40,Q42, D43, E44, K49, Y51, S54, N55, F56, S57, Q62, E101, N102, E124,E131, R142, D149, T150, E185, R192, D195, E201 and E203, such asmutations at one or more of the following positions N40, Q42, D43, E44,K49, Y51, S54, N55, F56, S57, Q62, E101, N102, E131, D149, T150, E185,D195, E201 and E203 or one or mutations at one or more of the followingpositions N40, Q42, D43, E44, K49, Y51, N55, F56, E101, N102, E131,D149, T150, E185, D195, E201 and E203; and/or (b) deletion of one ormore of the following positions F48, K49, P50, Y51, P52, A53, S54, N55,F56 and S57. The variant may comprise (a); (b); or (a) and (b). In (a),any number and combination of positions N40, Q42, D43, E44, K49, Y51,S54, N55, F56, S57, Q62, E101, N102, R124, E131, R142, D149, E185, R192,D195, E201 and E203 may be mutated. As discussed above, mutating one ormore of Y51, N55 and F56 may decrease the number of nucleotidescontributing to the current as the polynucleotide moves through the poreand thereby make it easier to identify a direct relationship between theobserved current as the polynucleotide moves through the pore and thepolynucleotide.

In (b), any number and combination of F48, K49, P50, Y51, P52, A53, S54,N55, F56 and S57 may be deleted. The variant may comprise any of thespecific mutations/substitutions or combinations thereof discussed abovewith reference to the mutant monomers of the invention.

Preferred variants for use in the method of the invention comprise oneor more of the following substitutions (a) F56N, F56Q, F56R, F56S, F56G,F56A or F56K or F56A, F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T orF56G; (b) N55Q, N55R, N55K, N55S, N55G, N55A or N55T; (c) Y51L, Y51V,Y51A, Y51N, Y51Q, Y51S or Y51G; (d) T150I; (e) S54P; and (f) S57P. Thevariant may comprise any number and combination of (a) to (f).

Preferred variants for use in the method of the invention comprise Q62Ror Q62K.

Preferred variants for use in the method of the invention comprisemutations at D43, E44, Q62 or any combination thereof, such as D43, E44,Q62, D43/E44, D43/Q62, E44/Q62 or D43/E44/Q62.

The variant may comprise a mutation at the following positions:

-   -   Y51, F56, D149, E185, E201 and E203, such as Y51N, F56A, D149N,        E185R, E201N and E203N;    -   N55, such as N55A or N55S;    -   Y51, such as Y51N or Y51T;    -   S54, such as S54P;    -   S57, such as S57P;    -   F56, such as F56N, F56Q, F56R, F56S, F56G, F56A or F56K or F56A,        F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T or F56G;    -   Y51 and F56, such as Y51A and F56A, Y51A and F56N, Y51I and        F56A, Y51L and F56A, Y51T and F56A, Y51T and F56Q, Y51I and        F56N, Y51L and F56N or Y51T and F56N, preferably, Y51I and F56A,        Y51L and F56A or Y51T and F56A, more preferably Y51T and F56Q or        more preferably Y51X and F56Q, wherein X is any amino acid;    -   N55 and F56, such as N55X and F56Q, wherein X is any amino acid;    -   Y51, N55 and F56, such as Y51A, N55S and F56A, Y51A, N55S and        F56N or Y51T, N55S and F56Q;    -   S54 and F56, such as S54P and F56A or S54P and F56N;    -   F56 and S57, such as F56A and S57P or F56N and S57P;    -   D149, E185 and E203, such as D149N, E185N and E203N;    -   D149, E185, E201 and E203, such as D149N, E185N, E201N and        E203N;    -   D149, E185, D195, E201 and E203, such as D149N, E185R, D195N,        E201N and E203N or D149N, E185R, D195N, E201R and E203N;    -   F56 and N102, such as F56Q and N102R;    -   (a) Q62, such as Q62R or Q62K, and (b) one or more of Y51, N55        and F56, such as at Y51, N55, F56, Y51/N55, Y51/F56, N55/F56 or        Y51/N55/F56, such as Y51T/F56Q/Q62R;    -   (i) D43, E44, Q62 or any combination thereof, such as D43, E44,        Q62, D43/E44, D43/Q62, E44/Q62 or D43/E44/Q62 and (ii) one or        more of Y51, N55 and F56, such as at Y51, N55, F56, Y51/N55,        Y51/F56, N55/F56 or Y51/N55/F56, such as D43N/Y51T/F56Q,        E44N/Y51T/F56Q, D43N/E44N/Y51T/F56Q, D43N/Y51T/F56Q/Q62R,        E44N/Y51T/F56Q/Q62R or D43N/E44N/Y51T/F56Q/Q62R; or    -   T150, such as T150I.

Preferred pores for use in the method of the invention comprise at least7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10monomers, each of which comprises a variant of SEQ ID NO: 390 whichcomprises one or more of the following substitutions (a) F56N, F56Q,F56R, F56S, F56G, F56A or F56K or F56A, F56P, F56R, F56H, F56S, F56Q,F56I, F56L, F56T or F56G; (b) N55Q, N55R, N55K, N55S, N55G, N55A orN55T; (c) Y51L, Y51V, Y51A, Y51N, Y51Q, Y51S or Y51G; (d) T150I; (e)S54P; and (f) S57P. The variants may comprise any number and combinationof (a) to (f). The monomers are preferably identical in these preferredpores.

Preferred pores for use in the method of the invention comprise at least7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10monomers, each of which comprises a variant of SEQ ID NO: 390 whichcomprises:

-   -   Y51N, F56A, D149N, E185R, E201N and E203N;    -   N55A;    -   N55S;    -   Y51N;    -   S54P;    -   S57P;    -   F56N, F56Q, F56R, F56S, F56G, F56A or F56K;    -   F56A, F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T or F56G;    -   Y51A and F56A;    -   Y51A and F56N;    -   Y51I and F56A;    -   Y51L and F56A;    -   Y51T and F56A;    -   Y51T and F56Q;    -   Y51I and F56N;    -   Y51L and F56N;    -   Y51T and F56N;    -   N55S and F56Q;    -   Y51A, N55S and F56A;    -   Y51A, N55S and F56N;    -   Y51T, N55S and F56Q;    -   S54P and F56A;    -   S54P and F56N;    -   F56A and S57P;    -   F56N and S57P;    -   D149N, E185N and E203N;    -   D149N, E185N, E201N and E203N;    -   D149N, E185R, D195N, E201N and E203N;    -   D149N, E185R, D195N, E201R and E203N;    -   T150I;    -   F56Q and N102R;    -   F56 and N102, such as F56Q and N102R;    -   Y51T/F56Q/Q62R;    -   D43N/Y51T/F56Q;    -   E44N/Y51T/F56Q;    -   D43N/E44N/Y51T/F56Q    -   D43N/Y51T/F56Q/Q62R;    -   E44N/Y51T/F56Q/Q62R; or    -   D43 N/E44N/Y51T/F56Q/Q62R.

The CsgG mutant for use in the method of the invention preferablycomprises nine monomers and at least one of the monomers is a variant ofthe sequence shown in SEQ ID NO: 390 comprising a mutation at one ormore of positions Y51, N55 and F56. Preferred pores for use in themethod of the invention comprise at least 7, at least 8, at least 9 orat least 10 monomers, such as 7, 8, 9 or 10 monomers, each of whichcomprises a variant of SEQ ID NO: 390 comprising a mutation at one ormore of positions Y51, N55 and F56. The monomers are preferablyidentical in these preferred pores. The variant may comprise a mutationat Y51; N55; F56; Y51/N55; Y51/F56; N55/F56; or Y51/N55/F56. The variantmay comprise any of the specific mutations at one or more of positionsY51, N55 and F56 discussed above and in any combination. One or moreY51, N55 and F56 may be substituted with any amino acid. Y51 may besubstituted with F, M, L, I, V, A, P, G, C, Q, N, T, S, E, D, K, H or R,such as A, S, T, N or Q. N55 may be substituted with F, M, L, I, V, A,P, G, C, Q, T, S, E, D, K, H or R, such as A, S, T or Q. F56 may besubstituted with M, L, I, V, A, P, G, C, Q, N, T, S, E, D, K, H or R,such as A, S, T, N or Q. The variant may further comprise one or more ofthe following: (i) one or more mutations at the following positions(i.e. mutations at one or more of the following positions) (i) N40, D43,E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150 and R192; (iii)Q42R or Q42K; (iv) K49R; (v) N102R, N102F, N102Y or N102W; (vi) D149N,D149Q or D149R; (vii) E185N, E185Q or E185R; (viii) D195N, D195Q orD195R; (ix) E201N, E201Q or E201R; (x) E203N, E203Q or E203R; and (xi)deletion of one or more of the following positions F48, K49, P50, Y51,P52, A53, S54, N55, F56 and S57. The variant may comprise any of thecombinations of (i) and (ii) to (xi) discussed above. The variant maycomprise any of the embodiments discussed above for (i) and (iii) to(xi).

-   -   (1) Preferred variants for use in the method of the invention        comprise or (2) preferred pores for use in the method of the        invention comprise at least 7, at least 8, at least 9 or at        least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which        comprises a variant of SEQ ID NO: 390 which comprise: Y51R/F56Q,        Y51N/F56N, Y51M/F56Q, Y51L/F56Q, Y51/F56Q, Y51V/F56Q, Y51A/F56Q,        Y51P/F56Q, Y51G/F56Q, F51C/F56Q, Y51Q/F56Q, Y51N/F56Q,        Y51S/F56Q, Y51E/F56Q, Y51D/F56Q, Y51K/F56Q or Y51H/F56Q.    -   (1) Preferred variants for use in the method of the invention        comprise or (2) preferred pores for use in the method of the        invention comprise at least 7, at least 8, at least 9 or at        least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which        comprises a variant of SEQ ID NO: 390 which comprise: Y51T/F56Q,        Y51Q/F56Q or Y51A/F56Q.    -   (1) Preferred variants for use in the method of the invention        comprise or (2) preferred pores for use in the method of the        invention comprise at least 7, at least 8, at least 9 or at        least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which        comprises a variant of SEQ ID NO: 390 which comprise: Y51T/F56F,        Y51T/F56M, Y51T/F56L, Y51T/F56I, Y51T/F56V, Y51T/F56A,        Y51T/F56P, Y51T/F56G, Y51T/F56C, Y51T/F56Q, Y51T/F56N,        Y51T/F56T, Y51T/F56S, Y51T/F56E, Y51T/F56D, Y51T/F56K, Y51T/F56H        or Y51T/F56R.    -   (1) Preferred variants for use in the method of the invention        comprise or (2) preferred pores for use in the method of the        invention comprise at least 7, at least 8, at least 9 or at        least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which        comprises a variant of SEQ ID NO: 390 which comprise: Y51T/N55Q,        Y51T/N55S or Y51T/N55A.    -   (1) Preferred variants for use in the method of the invention        comprise or (2) preferred pores for use in the method of the        invention comprise at least 7, at least 8, at least 9 or at        least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which        comprises a variant of SEQ ID NO: 390 which comprise: Y51A/F56F,        Y51A/F56L, Y51A/F56I, Y51A/F56V, Y51A/F56A, Y51A/F56P,        Y51A/F56G, Y51A/F56C, Y51A/F56Q, Y51A/F56N, Y51A/F56T,        Y51A/F56S, Y51A/F56E, Y51A/F56D, Y51A/F56K, Y51A/F56H or        Y51A/F56R.    -   (1) Preferred variants for use in the method of the invention        comprise or (2) preferred pores for use in the method of the        invention comprise at least 7, at least 8, at least 9 or at        least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which        comprises a variant of SEQ ID NO: 390 which comprise: Y51C/F56A,        Y51E/F56A, Y51D/F56A, Y51K/F56A, Y51H/F56A, Y51Q/F56A,        Y51N/F56A, Y51S/F56A, Y51P/F56A or Y51V/F56A.    -   (1) Preferred variants for use in the method of the invention        comprise or (2) preferred pores for use in the method of the        invention comprise at least 7, at least 8, at least 9 or at        least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which        comprises a variant of SEQ ID NO: 390 which comprise:    -   D149R/E185R/E201R/E203R or Y51T/F56Q/D149R/E185R/E201R/E203R;    -   D149N/E185N/E201N/E203N or Y51T/F56Q/D149N/E185N/E201N/E203N;    -   E201R/E203R or Y51T/F56Q/E201R/E203R    -   E201N/E203R or Y51T/F56Q/E201N/E203R;    -   E203R or Y51T/F56Q/E203R;    -   E203N or Y51T/F56Q/E203N;    -   E201R or Y51T/F56Q/E201R;    -   E201N or Y51T/F56Q/E201N;    -   E185R or Y51T/F56Q/E185R;    -   E185N or Y51T/F56Q/E185N;    -   D149R or Y51T/F56Q/D149R;    -   D149N or Y51T/F56Q/D149N;    -   R142E or Y51T/F56Q/R142E;    -   R142N or Y51T/F56Q/R142N;    -   R192E or Y51T/F56Q/R192E; or    -   R192N or Y51T/F56Q/R192N.    -   (1) Preferred variants for use in the method of the invention        comprise or (2) preferred pores for use in the method of the        invention comprise at least 7, at least 8, at least 9 or at        least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which        comprises a variant of SEQ ID NO: 390 which comprise:    -   Y51A/F56Q/E101N/N102R;    -   Y51A/F56Q/R97N/N102G;    -   Y51A/F56Q/R97N/N102R;    -   Y51A/F56Q/R97N;    -   Y51A/F56Q/R97G;    -   Y51A/F56Q/R97L;    -   Y51A/F56Q/N102R;    -   Y51A/F56Q/N102F;    -   Y51A/F56Q/N102G;    -   Y51A/F56Q/E101R;    -   Y51A/F56Q/E101F;    -   Y51A/F56Q/E101N; or    -   Y51A/F56Q/E101G.

The monomers are preferably identical in these preferred pores.

Preferred pores for use in the method of the invention comprise at least7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10monomers, each of which comprises a variant of SEQ ID NO: 390 whichcomprises F56A, F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T or F56G.The monomers are preferably identical in these preferred pores.

The CsgG mutant may comprise any of the variants of SEQ ID NO: 390disclosed in the Examples or may be any of the pores disclosed in theExamples.

The CsgG mutant is most preferably a pore of the invention.

Steps (a) and (b) are preferably carried out with a potential appliedacross the pore. As discussed in more detail below, the appliedpotential typically results in the formation of a complex between thepore and a polynucleotide binding protein. The applied potential may bea voltage potential. Alternatively, the applied potential may be achemical potential. An example of this is using a salt gradient acrossan amphiphilic layer. A salt gradient is disclosed in Holden et al., JAm Chem Soc. 2007 Jul. 11; 129(27):8650-5.

The method is for determining the presence, absence or one or morecharacteristics of a target analyte. The method may be for determiningthe presence, absence or one or more characteristics of at least oneanalyte. The method may concern determining the presence, absence or oneor more characteristics of two or more analytes. The method may comprisedetermining the presence, absence or one or more characteristics of anynumber of analytes, such as 2, 5, 10, 15, 20, 30, 40, 50, 100 or moreanalytes. Any number of characteristics of the one or more analytes maybe determined, such as 1, 2, 3, 4, 5, 10 or more characteristics.

The target analyte is preferably a metal ion, an inorganic salt, apolymer, an amino acid, a peptide, a polypeptide, a protein, anucleotide, an oligonucleotide, a polynucleotide, a dye, a bleach, apharmaceutical, a diagnostic agent, a recreational drug, an explosive oran environmental pollutant. The method may concern determining thepresence, absence or one or more characteristics of two or more analytesof the same type, such as two or more proteins, two or more nucleotidesor two or more pharmaceuticals. Alternatively, the method may concerndetermining the presence, absence or one or more characteristics of twoor more analytes of different types, such as one or more proteins, oneor more nucleotides and one or more pharmaceuticals.

The target analyte can be secreted from cells. Alternatively, the targetanalyte can be an analyte that is present inside cells such that theanalyte must be extracted from the cells before the invention can becarried out.

The analyte is preferably an amino acid, a peptide, a polypeptidesand/or a protein. The amino acid, peptide, polypeptide or protein can benaturally-occurring or non-naturally-occurring. The polypeptide orprotein can include within them synthetic or modified amino acids. Anumber of different types of modification to amino acids are known inthe art. Suitable amino acids and modifications thereof are above. Forthe purposes of the invention, it is to be understood that the targetanalyte can be modified by any method available in the art.

The protein can be an enzyme, an antibody, a hormone, a growth factor ora growth regulatory protein, such as a cytokine. The cytokine may beselected from interleukins, preferably IFN-1, IL-1, IL-2, IL-4, IL-5,IL-6, IL-10, IL-12 and IL-13, interferons, preferably IL-γ, and othercytokines such as TNF-α. The protein may be a bacterial protein, afungal protein, a virus protein or a parasite-derived protein.

The target analyte is preferably a nucleotide, an oligonucleotide or apolynucleotide. Nucleotides and polynucleotides are discussed below.Oligonucleotides are short nucleotide polymers which typically have 50or fewer nucleotides, such 40 or fewer, 30 or fewer, 20 or fewer, 10 orfewer or 5 or fewer nucleotides. The oligonucleotides may comprise anyof the nucleotides discussed below, including the abasic and modifiednucleotides.

The target analyte, such as a target polynucleotide, may be present inany of the suitable samples discussed below.

The pore is typically present in a membrane as discussed below. Thetarget analyte may be coupled or delivered to the membrane using of themethods discussed below.

Any of the measurements discussed below can be used to determine thepresence, absence or one or more characteristics of the target analyte.The method preferably comprises contacting the target analyte with thepore such that the analyte moves with respect to, such as moves through,the pore and measuring the current passing through the pore as theanalyte moves with respect to the pore and thereby determining thepresence, absence or one or more characteristics of the analyte.

The target analyte is present if the current flows through the pore in amanner specific for the analyte (i.e. if a distinctive currentassociated with the analyte is detected flowing through the pore). Theanalyte is absent if the current does not flow through the pore in amanner specific for the nucleotide. Control experiments can be carriedout in the presence of the analyte to determine the way in which ifaffects the current flowing through the pore.

The invention can be used to differentiate analytes of similar structureon the basis of the different effects they have on the current passingthrough a pore. Individual analytes can be identified at the singlemolecule level from their current amplitude when they interact with thepore. The invention can also be used to determine whether or not aparticular analyte is present in a sample. The invention can also beused to measure the concentration of a particular analyte in a sample.Analyte characterisation using pores other than CsgG is known in theart.

Polynucleotide Characterisation

The invention provides a method of characterising a targetpolynucleotide, such as sequencing a polynucleotide. There are two mainstrategies for characterising or sequencing polynucleotides usingnanopores, namely strand characterisation/sequencing and exonucleasecharacterisation/sequencing. The method of the invention may concerneither method.

In strand sequencing, the DNA is translocated through the nanoporeeither with or against an applied potential. Exonucleases that actprogressively or processively on double stranded DNA can be used on thecis side of the pore to feed the remaining single strand through underan applied potential or the trans side under a reverse potential.Likewise, a helicase that unwinds the double stranded DNA can also beused in a similar manner. A polymerase may also be used. There are alsopossibilities for sequencing applications that require strandtranslocation against an applied potential, but the DNA must be first“caught” by the enzyme under a reverse or no potential. With thepotential then switched back following binding the strand will pass cisto trans through the pore and be held in an extended conformation by thecurrent flow. The single strand DNA exonucleases or single strand DNAdependent polymerases can act as molecular motors to pull the recentlytranslocated single strand beck through the pore in a controlledstepwise manner, trans to cis, against the applied potential.

In one embodiment, the method of characterising a target polynucleotideinvolves contacting the target sequence with a pore and a helicaseenzyme. Any helicase may be used in the method. Suitable helicases arediscussed below. Helicases may work in two modes with respect to thepore. First, the method is preferably carried out using a helicase suchthat it controls movement of the target sequence through the pore withthe field resulting from the applied voltage. In this mode the 5′ end ofthe DNA is first captured in the pore, and the enzyme controls movementof the DNA into the pore such that the target sequence is passed throughthe pore with the field until it finally translocates through to thetrans side of the bilayer. Alternatively, the method is preferablycarried out such that a helicase enzyme controls movement of the targetsequence through the pore against the field resulting from the appliedvoltage. In this mode the 3′ end of the DNA is first captured in thepore, and the enzyme controls movement of the DNA through the pore suchthat the target sequence is pulled out of the pore against the appliedfield until finally ejected back to the cis side of the bilayer.

In exonuclease sequencing, an exonuclease releases individualnucleotides from one end of the target polynucleotide and theseindividual nucleotides are identified as discussed below. In anotherembodiment, the method of characterising a target polynucleotideinvolves contacting the target sequence with a pore and an exonucleaseenzyme. Any of the exonuclease enzymes discussed below may be used inthe method. The enzyme may be covalently attached to the pore asdiscussed below.

Exonucleases are enzymes that typically latch onto one end of apolynucleotide and digest the sequence one nucleotide at a time fromthat end. The exonuclease can digest the polynucleotide in the 5′ to 3′direction or 3′ to 5′ direction. The end of the polynucleotide to whichthe exonuclease binds is typically determined through the choice ofenzyme used and/or using methods known in the art. Hydroxyl groups orcap structures at either end of the polynucleotide may typically be usedto prevent or facilitate the binding of the exonuclease to a particularend of the polynucleotide.

The method involves contacting the polynucleotide with the exonucleaseso that the nucleotides are digested from the end of the polynucleotideat a rate that allows characterisation or identification of a proportionof nucleotides as discussed above. Methods for doing this are well knownin the art. For example, Edman degradation is used to successivelydigest single amino acids from the end of polypeptide such that they maybe identified using High Performance Liquid Chromatography (HPLC). Ahomologous method may be used in the present invention.

The rate at which the exonuclease functions is typically slower than theoptimal rate of a wild-type exonuclease. A suitable rate of activity ofthe exonuclease in the method of the invention involves digestion offrom 0.5 to 1000 nucleotides per second, from 0.6 to 500 nucleotides persecond, 0.7 to 200 nucleotides per second, from 0.8 to 100 nucleotidesper second, from 0.9 to 50 nucleotides per second or 1 to 20 or 10nucleotides per second. The rate is preferably 1, 10, 100, 500 or 1000nucleotides per second. A suitable rate of exonuclease activity can beachieved in various ways. For example, variant exonucleases with areduced optimal rate of activity may be used in accordance with theinvention.

In the strand characterisation embodiment, the method comprisescontacting the polynucleotide with a CsgG pore or mutant thereof, suchas a pore of the invention, such that the polynucleotide moves withrespect to, such as through, the pore and taking one or moremeasurements as the polynucleotide moves with respect to the pore,wherein the measurements are indicative of one or more characteristicsof the polynucleotide, and thereby characterising the targetpolynucleotide.

In the exonucleotide characterisation embodiment, the method comprisescontacting the polynucleotide with a CsgG pore or mutant thereof, suchas a pore of the invention, and an exonuclease such that the exonucleasedigests individual nucleotides from one end of the target polynucleotideand the individual nucleotides move with respect to, such as through,the pore and taking one or more measurements as the individualnucleotides move with respect to the pore, wherein the measurements areindicative of one or more characteristics of the individual nucleotides,and thereby characterising the target polynucleotide.

An individual nucleotide is a single nucleotide. An individualnucleotide is one which is not bound to another nucleotide orpolynucleotide by a nucleotide bond. A nucleotide bond involves one ofthe phosphate groups of a nucleotide being bound to the sugar group ofanother nucleotide. An individual nucleotide is typically one which isnot bound by a nucleotide bond to another polynucleotide of at least 5,at least 10, at least 20, at least 50, at least 100, at least 200, atleast 500, at least 1000 or at least 5000 nucleotides. For example, theindividual nucleotide has been digested from a target polynucleotidesequence, such as a DNA or RNA strand. The nucleotide can be any ofthose discussed below.

The individual nucleotides may interact with the pore in any manner andat any site. The nucleotides preferably reversibly bind to the pore viaor in conjunction with an adaptor as discussed above. The nucleotidesmost preferably reversibly bind to the pore via or in conjunction withthe adaptor as they pass through the pore across the membrane. Thenucleotides can also reversibly bind to the barrel or channel of thepore via or in conjunction with the adaptor as they pass through thepore across the membrane.

During the interaction between the individual nucleotide and the pore,the nucleotide typically affects the current flowing through the pore ina manner specific for that nucleotide. For example, a particularnucleotide will reduce the current flowing through the pore for aparticular mean time period and to a particular extent. In other words,the current flowing through the pore is distinctive for a particularnucleotide. Control experiments may be carried out to determine theeffect a particular nucleotide has on the current flowing through thepore. Results from carrying out the method of the invention on a testsample can then be compared with those derived from such a controlexperiment in order to identify a particular nucleotide in the sample ordetermine whether a particular nucleotide is present in the sample. Thefrequency at which the current flowing through the pore is affected in amanner indicative of a particular nucleotide can be used to determinethe concentration of that nucleotide in the sample. The ratio ofdifferent nucleotides within a sample can also be calculated. Forinstance, the ratio of dCMP to methyl-dCMP can be calculated.

The method involves measuring one or more characteristics of the targetpolynucleotide. The target polynucleotide may also be called thetemplate polynucleotide or the polynucleotide of interest.

This embodiment also uses a CsgG pore or mutant thereof, such as a poreof the invention. Any of the pores and embodiments discussed above withreference to the target analyte may be used.

Polynucleotide

A polynucleotide, such as a nucleic acid, is a macromolecule comprisingtwo or more nucleotides. The polynucleotide or nucleic acid may compriseany combination of any nucleotides. The nucleotides can be naturallyoccurring or artificial. One or more nucleotides in the polynucleotidecan be oxidized or methylated. One or more nucleotides in thepolynucleotide may be damaged. For instance, the polynucleotide maycomprise a pyrimidine dimer. Such dimers are typically associated withdamage by ultraviolet light and are the primary cause of skin melanomas.One or more nucleotides in the polynucleotide may be modified, forinstance with a label or a tag. Suitable labels are described below. Thepolynucleotide may comprise one or more spacers.

A nucleotide typically contains a nucleobase, a sugar and at least onephosphate group. The nucleobase and sugar form a nucleoside.

The nucleobase is typically heterocyclic. Nucleobases include, but arenot limited to, purines and pyrimidines and more specifically adenine(A), guanine (G), thymine (T), uracil (U) and cytosine (C).

The sugar is typically a pentose sugar. Nucleotide sugars include, butare not limited to, ribose and deoxyribose. The sugar is preferably adeoxyribose.

The polynucleotide preferably comprises the following nucleosides:deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT),deoxyguanosine (dG) and deoxycytidine (dC).

The nucleotide is typically a ribonucleotide or deoxyribonucleotide. Thenucleotide typically contains a monophosphate, diphosphate ortriphosphate. The nucleotide may comprise more than three phosphates,such as 4 or 5 phosphates. Phosphates may be attached on the 5′ or 3′side of a nucleotide. Nucleotides include, but are not limited to,adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidinemonophosphate (TMP), uridlne monophosphate (UMP), 5-methylcytidinemonophosphate, 5-hydroxymethylcytidine monophosphate, cytidinemonophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclicguanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate(dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate(dCMP) and deoxymethylcytidine monophosphate. The nucleotides arepreferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMPand dUMP.

A nucleotide may be abasic (i.e. lack a nucleobase). A nucleotide mayalso lack a nucleobase and a sugar (i.e. is a C3 spacer).

The nucleotides in the polynucleotide may be attached to each other inany manner. The nucleotides are typically attached by their sugar andphosphate groups as in nucleic acids. The nucleotides may be connectedvia their nucleobases as in pyrimidine dimers.

The polynucleotide may be single stranded or double stranded. At least aportion of the polynucleotide is preferably double stranded.

The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid(DNA) or ribonucleic acid (RNA). The polynucleotide can comprise onestrand of RNA hybridised to one strand of DNA. The polynucleotide may beany synthetic nucleic acid known in the art, such as peptide nucleicacid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA),locked nucleic acid (LNA) or other synthetic polymers with nucleotideside chains. The PNA backbone is composed of repeatingN-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backboneis composed of repeating glycol units linked by phosphodiester bonds.The TNA backbone is composed of repeating threose sugars linked togetherby phosphodiester bonds. LNA is formed from ribonucleotides as discussedabove having an extra bridge connecting the 2′ oxygen and 4′ carbon inthe ribose moiety. Bridged nucleic acids (BNAs) are modified RNAnucleotides. They may also be called constrained or inaccessible RNA.BNA monomers can contain a five-membered, six-membered or even aseven-membered bridged structure with a “fixed” C3′-endo sugarpuckering. The bridge is synthetically incorporated at the 2′,4′-position of the ribose to produce a 2′, 4′-BNA monomer.

The polynucleotide is most preferably ribonucleic nucleic acid (RNA) ordeoxyribonucleic acid (DNA).

The polynucleotide can be any length. For example, the polynucleotidecan be at least 10, at least 50, at least 100, at least 150, at least200, at least 250, at least 300, at least 400 or at least 500nucleotides or nucleotide pairs in length. The polynucleotide can be1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotidesor nucleotide pairs in length or 100000 or more nucleotides ornucleotide pairs in length.

Any number of polynucleotides can be investigated. For instance, themethod of the invention may concern characterising 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 30, 50, 100 or more polynucleotides. If two or morepolynucleotides are characterised, they may be different polynucleotidesor two instances of the same polynucleotide.

The polynucleotide can be naturally occurring or artificial. Forinstance, the method may be used to verify the sequence of amanufactured oligonucleotide. The method is typically carried out invitro.

Sample

The polynucleotide is typically present in any suitable sample. Theinvention is typically carried out on a sample that is known to containor suspected to contain the polynucleotide. Alternatively, the inventionmay be carried out on a sample to confirm the identity of apolynucleotide whose presence in the sample is known or expected.

The sample may be a biological sample. The invention may be carried outin vitro using a sample obtained from or extracted from any organism ormicroorganism. The organism or microorganism is typically archaeal,prokaryotic or eukaryotic and typically belongs to one of the fivekingdoms: plantae, animalia, fungi, monera and protista. The inventionmay be carried out in vitro on a sample obtained from or extracted fromany virus. The sample is preferably a fluid sample. The sample typicallycomprises a body fluid of the patient. The sample may be urine, lymph,saliva, mucus or amniotic fluid but is preferably blood, plasma orserum.

Typically, the sample is human in origin, but alternatively it may befrom another mammal animal such as from commercially farmed animals suchas horses, cattle, sheep, fish, chickens or pigs or may alternatively bepets such as cats or dogs. Alternatively, the sample may be of plantorigin, such as a sample obtained from a commercial crop, such as acereal, legume, fruit or vegetable, for example wheat, barley, oats,canola, maize, soya, rice, rhubarb, bananas, apples, tomatoes, potatoes,grapes, tobacco, beans, lentils, sugar cane, cocoa, cotton.

The sample may be a non-biological sample. The non-biological sample ispreferably a fluid sample. Examples of non-biological samples includesurgical fluids, water such as drinking water, sea water or river water,and reagents for laboratory tests.

The sample is typically processed prior to being used in the invention,for example by centrifugation or by passage through a membrane thatfilters out unwanted molecules or cells, such as red blood cells. Themay be measured immediately upon being taken. The sample may also betypically stored prior to assay, preferably below −70° C.

Characterisation

The method may involve measuring two, three, four or five or morecharacteristics of the polynucleotide. The one or more characteristicsare preferably selected from (i) the length of the polynucleotide, (ii)the identity of the polynucleotide, (iii) the sequence of thepolynucleotide, (iv) the secondary structure of the polynucleotide and(v) whether or not the polynucleotide is modified. Any combination of(i) to (v) may be measured in accordance with the invention, such as{i}, {ii}, {iii}, {iv}, {v}, {i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii},{ii,iv}, {ii,v}, {iii,iv}, {iii,v}, {iv,v}, {i,ii,iii}, {i,ii,iv},{i,ii,v}, {i,iii,iv}, {i,iii,v}, {i,iv,v}, {i,iii,iv}, {ii,iii,v},{ii,iv,v}, {iii,iv,v}, {i,ii,iii,iv}, {i,ii,iii,v}, {i,ii,iv,v},{i,iii,iv,v}, {ii,iii,iv,v} or {i,ii,iii,iv,v}. Different combinationsof (i) to (v) may be measured for the first polynucleotide compared withthe second polynucleotide, including any of those combinations listedabove.

For (i), the length of the polynucleotide may be measured for example bydetermining the number of interactions between the polynucleotide andthe pore or the duration of interaction between the polynucleotide andthe pore.

For (ii), the identity of the polynucleotide may be measured in a numberof ways. The identity of the polynucleotide may be measured inconjunction with measurement of the sequence of the polynucleotide orwithout measurement of the sequence of the polynucleotide. The former isstraightforward; the polynucleotide is sequenced and thereby identified.The latter may be done in several ways. For instance, the presence of aparticular motif in the polynucleotide may be measured (withoutmeasuring the remaining sequence of the polynucleotide). Alternatively,the measurement of a particular electrical and/or optical signal in themethod may identify the polynucleotide as coming from a particularsource.

For (iii), the sequence of the polynucleotide can be determined asdescribed previously. Suitable sequencing methods, particularly thoseusing electrical measurements, are described in Stoddart D et al., ProcNatl Acad Sci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc.2010; 132(50):17961-72, and International Application WO 2000/28312.

For (iv), the secondary structure may be measured in a variety of ways.For instance, if the method involves an electrical measurement, thesecondary structure may be measured using a change in dwell time or achange in current flowing through the pore. This allows regions ofsingle-stranded and double-stranded polynucleotide to be distinguished.

For (v), the presence or absence of any modification may be measured.The method preferably comprises determining whether or not thepolynucleotide is modified by methylation, by oxidation, by damage, withone or more proteins or with one or more labels, tags or spacers.Specific modifications will result in specific interactions with thepore which can be measured using the methods described below. Forinstance, methylcytosine may be distinguished from cytosine on the basisof the current flowing through the pore during its interaction with eachnucleotide.

The target polynucleotide is contacted with a CsgG pore or mutantthereof, such as a pore of the invention. The pore is typically presentin a membrane. Suitable membranes are discussed below. The method may becarried out using any apparatus that is suitable for investigating amembrane/pore system in which a pore is present in a membrane. Themethod may be carried out using any apparatus that is suitable fortransmembrane pore sensing. For example, the apparatus comprises achamber comprising an aqueous solution and a barrier that separates thechamber into two sections. The barrier typically has an aperture inwhich the membrane containing the pore is formed. Alternatively thebarrier forms the membrane in which the pore is present.

The method may be carried out using the apparatus described inInternational Application No. PCT/GB08/000562 (WO 2008/102120).

A variety of different types of measurements may be made. This includeswithout limitation: electrical measurements and optical measurements.Possible electrical measurements include: current measurements,impedance measurements, tunneling measurements (Ivanov A P et al., NanoLett. 2011 Jan. 12; 11(1):279-85), and FET measurements (InternationalApplication WO 2005/124888). Optical measurements may be combined withelectrical measurements (Soni G V et al., Rev Sci Instrum. 2010 January;81(1):014301). The measurement may be a transmembrane currentmeasurement such as measurement of ionic current flowing through thepore.

Electrical measurements may be made using standard single channelrecording equipment as describe in Stoddart D et al., Proc Natl AcadSci, 12; 106(19):7702-7, Lieberman K R et al, J Am Chem Soc. 2010;132(50):17961-72, and International Application WO 2000/28312.Alternatively, electrical measurements may be made using a multi-channelsystem, for example as described in International Application WO2009/077734 and International Application WO 2011/067559.

The method is preferably carried out with a potential applied across themembrane. The applied potential may be a voltage potential.Alternatively, the applied potential may be a chemical potential. Anexample of this is using a salt gradient across a membrane, such as anamphiphilic layer. A salt gradient is disclosed in Holden et al., J AmChem Soc. 2007 Jul. 11; 129(27):8650-5. In some instances, the currentpassing through the pore as a polynucleotide moves with respect to thepore is used to estimate or determine the sequence of thepolynucleotide. This is strand sequencing.

The method may involve measuring the current passing through the pore asthe polynucleotide moves with respect to the pore. Therefore theapparatus used in the method may also comprise an electrical circuitcapable of applying a potential and measuring an electrical signalacross the membrane and pore. The methods may be carried out using apatch clamp or a voltage clamp. The methods preferably involve the useof a voltage clamp.

The method of the invention may involve the measuring of a currentpassing through the pore as the polynucleotide moves with respect to thepore. Suitable conditions for measuring ionic currents throughtransmembrane protein pores are known in the art and disclosed in theExample. The method is typically carried out with a voltage appliedacross the membrane and pore. The voltage used is typically from +5 V to−5 V, such as from +4 V to −4 V, +3 V to −3 V or +2 V to −2 V. Thevoltage used is typically from −600 mV to +600 mV or −400 mV to +400 mV.The voltage used is preferably in a range having a lower limit selectedfrom −400 mV, −300 mV, −200 mV, −150 mV, −100 mV, −50 mV, −20 mV and 0mV and an upper limit independently selected from +10 mV, +20 mV, +50mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used ismore preferably in the range 100 mV to 240 mV and most preferably in therange of 120 mV to 220 mV. It is possible to increase discriminationbetween different nucleotides by a pore by using an increased appliedpotential.

The method is typically carried out in the presence of any chargecarriers, such as metal salts, for example alkali metal salt, halidesalts, for example chloride salts, such as alkali metal chloride salt.Charge carriers may include ionic liquids or organic salts, for exampletetramethyl ammonium chloride, trimethylphenyl ammonium chloride,phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazoliumchloride. In the exemplary apparatus discussed above, the salt ispresent in the aqueous solution in the chamber. Potassium chloride(KCl), sodium chloride (NaCl), caesium chloride (CsCl) or a mixture ofpotassium ferrocyanide and potassium ferricyanide is typically used.KCl, NaCl and a mixture of potassium ferrocyanide and potassiumferricyanide are preferred. The charge carriers may be asymmetric acrossthe membrane. For instance, the type and/or concentration of the chargecarriers may be different on each side of the membrane.

The salt concentration may be at saturation. The salt concentration maybe 3 M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M,from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to1.4 M. The salt concentration is preferably from 150 mM to 1 M. Themethod is preferably carried out using a salt concentration of at least0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M orat least 3.0 M. High salt concentrations provide a high signal to noiseratio and allow for currents indicative of the presence of a nucleotideto be identified against the background of normal current fluctuations.

The method is typically carried out in the presence of a buffer. In theexemplary apparatus discussed above, the buffer is present in theaqueous solution in the chamber. Any buffer may be used in the method ofthe invention. Typically, the buffer is phosphate buffer. Other suitablebuffers are HEPES and Tris-HCl buffer. The methods are typically carriedout at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5. The pHused is preferably about 7.5.

The method may be carried out at from 0° C. to 100° C., from 15° C. to95° C., from 16° C. to 90° C., from 17° C. to 85° C., from 18° C. to 80°C., 19° C. to 70° C., or from 20° C. to 60° C. The methods are typicallycarried out at room temperature. The methods are optionally carried outat a temperature that supports enzyme function, such as about 37° C.

Polynucleotide Binding Protein

The strand characterisation method preferably comprises contacting thepolynucleotide with a polynucleotide binding protein such that theprotein controls the movement of the polynucleotide with respect to,such as through, the pore.

More preferably, the method comprises (a) contacting the polynucleotidewith a CsgG pore or mutant thereof, such as a pore of the invention, anda polynucleotide binding protein such that the protein controls themovement of the polynucleotide with respect to, such as through, thepore and (b) taking one or more measurements as the polynucleotide moveswith respect to the pore, wherein the measurements are indicative of oneor more characteristics of the polynucleotide, and therebycharacterising the polynucleotide.

More preferably, the method comprises (a) contacting the polynucleotidewith a CsgG pore or mutant thereof, such as a pore of the invention, anda polynucleotide binding protein such that the protein controls themovement of the polynucleotide with respect to, such as through, thepore and (b) measuring the current through the pore as thepolynucleotide moves with respect to the pore, wherein the current isindicative of one or more characteristics of the polynucleotide, andthereby characterising the polynucleotide.

The polynucleotide binding protein may be any protein that is capable ofbinding to the polynucleotide and controlling its movement through thepore. It is straightforward in the art to determine whether or not aprotein binds to a polynucleotide. The protein typically interacts withand modifies at least one property of the polynucleotide. The proteinmay modify the polynucleotide by cleaving it to form individualnucleotides or shorter chains of nucleotides, such as di- ortrinucleotides. The protein may modify the polynucleotide by orientingit or moving it to a specific position, i.e. controlling its movement.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. A polynucleotide handling enzyme is apolypeptide that is capable of interacting with and modifying at leastone property of a polynucleotide. The enzyme may modify thepolynucleotide by cleaving it to form individual nucleotides or shorterchains of nucleotides, such as di- or trinucleotides. The enzyme maymodify the polynucleotide by orienting it or moving it to a specificposition. The polynucleotide handling enzyme does not need to displayenzymatic activity as long as it is capable of binding thepolynucleotide and controlling its movement through the pore. Forinstance, the enzyme may be modified to remove its enzymatic activity ormay be used under conditions which prevent it from acting as an enzyme.Such conditions are discussed in more detail below.

The polynucleotide handling enzyme is preferably derived from anucleolytic enzyme. The polynucleotide handling enzyme used in theconstruct of the enzyme is more preferably derived from a member of anyof the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15,3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. Theenzyme may be any of those disclosed in International Application No.PCT/GB10/000133 (published as WO 2010/086603).

Preferred enzymes are polymerases, exonucleases, helicases andtopoisomerases, such as gyrases. Suitable enzymes include, but are notlimited to, exonuclease I from E. coli (SEQ ID NO: 399), exonuclease IIIenzyme from E. coli (SEQ ID NO: 401), RecJ from T. thermophilus (SEQ IDNO: 403) and bacteriophage lambda exonuclease (SEQ ID NO: 405), TatDexonuclease and variants thereof. Three subunits comprising the sequenceshown in SEQ ID NO: 403 or a variant thereof interact to form a trimerexonuclease. These exonucleases can also be used in the exonucleasemethod of the invention. The polymerase may be PyroPhage® 3173 DNAPolymerase (which is commercially available from Lucigen® Corporation),SD Polymerase (commercially available from Bioron®) or variants thereof.The enzyme is preferably Phi29 DNA polymerase (SEQ ID NO: 397) or avariant thereof. The topoisomerase is preferably a member of any of theMoiety Classification (EC) groups 5.99.1.2 and 5.99.1.3.

The enzyme is most preferably derived from a helicase, such as Hel308Mbu (SEQ ID NO: 406), Hel308 Csy (SEQ ID NO: 407), Hel308 Tga (SEQ IDNO: 408), Hel308 Mhu (SEQ ID NO: 409), Tral Eco (SEQ ID NO: 410), XPDMbu (SEQ ID NO: 411) or a variant thereof. Any helicase may be used inthe invention. The helicase may be or be derived from a Hel308 helicase,a RecD helicase, such as Tral helicase or a TrwC helicase, a XPDhelicase or a Dda helicase. The helicase may be any of the helicases,modified helicases or helicase constructs disclosed in InternationalApplication Nos. PCT/GB2012/052579 (published as WO 2013/057495);PCT/GB2012/053274 (published as WO 2013/098562); PCT/GB2012/053273(published as WO2013098561); PCT/GB2013/051925 (published as WO2014/013260); PCT/GB2013/051924 (published as WO 2014/013259);PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736.

The helicase preferably comprises the sequence shown in SEQ ID NO: 413(Trwc Cba) or as variant thereof, the sequence shown in SEQ ID NO: 406(Hel308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO:412 (Dda) or a variant thereof. Variants may differ from the nativesequences in any of the ways discussed below for transmembrane pores. Apreferred variant of SEQ ID NO: 412 comprises (a) E94C and A360C or (b)E94C, A360C, C109A and C136A and then optionally (ΔM1)G1G2 (i.e.deletion of M1 and then addition G1 and G2).

Any number of helicases may be used in accordance with the invention.For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may beused. In some embodiments, different numbers of helicases may be used.

The method of the invention preferably comprises contacting thepolynucleotide with two or more helicases. The two or more helicases aretypically the same helicase. The two or more helicases may be differenthelicases.

The two or more helicases may be any combination of the helicasesmentioned above. The two or more helicases may be two or more Ddahelicases. The two or more helicases may be one or more Dda helicasesand one or more TrwC helicases. The two or more helicases may bedifferent variants of the same helicase.

The two or more helicases are preferably attached to one another. Thetwo or more helicases are more preferably covalently attached to oneanother. The helicases may be attached in any order and using anymethod. Preferred helicase constructs for use in the invention aredescribed in International Application Nos. PCT/GB2013/051925 (publishedas WO 2014/013260); PCT/GB2013/051924 (published as WO 2014/013259);PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736.

A variant of SEQ ID NOs: 397, 399, 401, 403, 405, 406, 407, 408, 409,410, 411, 412 or 413 is an enzyme that has an amino acid sequence whichvaries from that of SEQ ID NO: 397, 399, 401, 403, 405, 406, 407, 408,409, 410, 411, 412 or 413 and which retains polynucleotide bindingability. This can be measured using any method known in the art. Forinstance, the variant can be contacted with a polynucleotide and itsability to bind to and move along the polynucleotide can be measured.The variant may include modifications that facilitate binding of thepolynucleotide and/or facilitate its activity at high saltconcentrations and/or room temperature. Variants may be modified suchthat they bind polynucleotides (i.e. retain polynucleotide bindingability) but do not function as a helicase (i.e. do not move alongpolynucleotides when provided with all the necessary components tofacilitate movement, e.g. ATP and Mg²⁺). Such modifications are known inthe art. For instance, modification of the Mg²⁺ binding domain inhelicases typically results in variants which do not function ashelicases. These types of variants may act as molecular brakes (seebelow).

Over the entire length of the amino acid sequence of SEQ ID NO: 397,399, 401, 403, 405, 406, 407, 408, 409, 410, 411, 412 or 413, a variantwill preferably be at least 50% homologous to that sequence based onamino acid identity. More preferably, the variant polypeptide may be atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90% and more preferably at least 95%,97% or 99% homologous based on amino acid identity to the amino acidsequence of SEQ ID NO: 397, 399, 401, 403, 405, 406, 407, 408, 409, 410,411, 412 or 413 over the entire sequence. There may be at least 80%, forexample at least 85%, 90% or 95%, amino acid identity over a stretch of200 or more, for example 230, 250, 270, 280, 300, 400, 500, 600, 700,800, 900 or 1000 or more, contiguous amino acids (“hard homology”).Homology is determined as described above. The variant may differ fromthe wild-type sequence in any of the ways discussed above with referenceto SEQ ID NO: 390 above. The enzyme may be covalently attached to thepore. Any method may be used to covalently attach the enzyme to thepore.

A preferred molecular brake is TrwC Cbe-Q594A (SEQ ID NO: 413 with themutation Q594A). This variant does not function as a helicase (i.e.binds polynucleotides but does not move along them when provided withall the necessary components to facilitate movement, e.g. ATP and Mg²⁺).

In strand sequencing, the polynucleotide is translocated through thepore either with or against an applied potential. Exonucleases that actprogressively or processively on double stranded polynucleotides can beused on the cis side of the pore to feed the remaining single strandthrough under an applied potential or the trans side under a reversepotential. Likewise, a helicase that unwinds the double stranded DNA canalso be used in a similar manner. A polymerase may also be used. Thereare also possibilities for sequencing applications that require strandtranslocation against an applied potential, but the DNA must be first“caught” by the enzyme under a reverse or no potential. With thepotential then switched back following binding the strand will pass cisto trans through the pore and be held in an extended conformation by thecurrent flow. The single strand DNA exonucleases or single strand DNAdependent polymerases can act as molecular motors to pull the recentlytranslocated single strand back through the pore in a controlledstepwise manner, trans to cis, against the applied potential.

Any helicase may be used in the method. Helicases may work in two modeswith respect to the pore. First, the method is preferably carried outusing a helicase such that it moves the polynucleotide through the porewith the field resulting from the applied voltage. In this mode the 5′end of the polynucleotide is first captured in the pore, and thehelicase moves the polynucleotide into the pore such that it is passedthrough the pore with the field until it finally translocates through tothe trans side of the membrane. Alternatively, the method is preferablycarried out such that a helicase moves the polynucleotide through thepore against the field resulting from the applied voltage. In this modethe 3′ end of the polynucleotide is first captured in the pore, and thehelicase moves the polynucleotide through the pore such that it ispulled out of the pore against the applied field until finally ejectedback to the cis side of the membrane.

The method may also be carried out in the opposite direction. The 3′ endof the polynucleotide may be first captured in the pore and the helicasemay move the polynucleotide into the pore such that it is passed throughthe pore with the field until it finally translocates through to thetrans side of the membrane.

When the helicase is not provided with the necessary components tofacilitate movement or is modified to hinder or prevent its movement, itcan bind to the polynucleotide and act as a brake slowing the movementof the polynucleotide when it is pulled into the pore by the appliedfield. In the inactive mode, it does not matter whether thepolynucleotide is captured either 3′ or 5′ down, it is the applied fieldwhich pulls the polynucleotide into the pore towards the trans side withthe enzyme acting as a brake. When in the inactive mode, the movementcontrol of the polynucleotide by the helicase can be described in anumber of ways including ratcheting, sliding and braking. Helicasevariants which lack helicase activity can also be used in this way.

The polynucleotide may be contacted with the polynucleotide bindingprotein and the pore in any order. It is preferred that, when thepolynucleotide is contacted with the polynucleotide binding protein,such as a helicase, and the pore, the polynucleotide firstly forms acomplex with the protein. When the voltage is applied across the pore,the polynucleotide/protein complex then forms a complex with the poreand controls the movement of the polynucleotide through the pore.

Any steps in the method using a polynucleotide binding protein aretypically carried out in the presence of free nucleotides or freenucleotide analogues and an enzyme cofactor that facilitates the actionof the polynucleotide binding protein. The free nucleotides may be oneor more of any of the individual nucleotides discussed above. The freenucleotides include, but are not limited to, adenosine monophosphate(AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP),guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosinetriphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate(TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP),uridine diphosphate (UDP), uridine triphosphate (UTP), cytidinemonophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate(CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosinemonophosphate (cGMP), deoxyadenosine monophosphate (dAMP),deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP),deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP),deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP),deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP),deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP),deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP),deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP).The free nucleotides are preferably selected from AMP, TMP, GMP, CMP,UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferablyadenosine triphosphate (ATP). The enzyme cofactor is a factor thatallows the construct to function. The enzyme cofactor is preferably adivalent metal cation. The divalent metal cation is preferably Mg²⁺,Mn²⁺, Ca²⁺ or Co²⁺. The enzyme cofactor is most preferably Mg²⁺.

Helicase(s) and Molecular Brake(s)

In a preferred embodiment, the method comprises:

-   -   (a) providing the polynucleotide with one or more helicases and        one or more molecular brakes attached to the polynucleotide;    -   (b) contacting the polynucleotide with a CsgG pore or mutant        thereof, such as a pore of the invention, and applying a        potential across the pore such that the one or more helicases        and the one or more molecular brakes are brought together and        both control the movement of the polynucleotide with respect to,        such as through, the pore;    -   (c) taking one or more measurements as the polynucleotide moves        with respect to the pore wherein the measurements are indicative        of one or more characteristics of the polynucleotide and thereby        characterising the polynucleotide.

This type of method is discussed in detail in the InternationalApplication PCT/GB2014/052737

The one or more helicases may be any of those discussed above. The oneor more molecular brakes may be any compound or molecule which binds tothe polynucleotide and slows the movement of the polynucleotide throughthe pore. The one or more molecular brakes preferably comprise one ormore compounds which bind to the polynucleotide. The one or morecompounds are preferably one or more macrocycles. Suitable macrocyclesinclude, but are not limited to, cyclodextrins, calixarenes, cyclicpeptides, crown ethers, cucurbiturils, pillararenes, derivatives thereofor a combination thereof. The cyclodextrin or derivative thereof may beany of those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J.Am. Chem. Soc. 116, 6081-6088. The agent is more preferablyheptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-β-cyclodextrin (am₁-□CD) orheptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu₇-βCD).

The one or more molecular brakes are preferably one or more singlestranded binding proteins (SSB). The one or more molecular brakes aremore preferably a single-stranded binding protein (SSB) comprising acarboxy-terminal (C-terminal) region which does not have a net negativecharge or (ii) a modified SSB comprising one or more modifications inits C-terminal region which decreases the net negative charge of theC-terminal region. The one or more molecular brakes are most preferablyone of the SSBs disclosed in International Application No.PCT/GB2013/051924 (published as WO 2014/013259).

The one or more molecular brakes are preferably one or morepolynucleotide binding proteins. The polynucleotide binding protein maybe any protein that is capable of binding to the polynucleotide andcontrolling its movement through the pore. It is straightforward in theart to determine whether or not a protein binds to a polynucleotide. Theprotein typically interacts with and modifies at least one property ofthe polynucleotide. The protein may modify the polynucleotide bycleaving it to form individual nucleotides or shorter chains ofnucleotides, such as di- or trinucleotides. The moiety may modify thepolynucleotide by orienting it or moving it to a specific position, i.e.controlling its movement.

The polynucleotide binding protein is preferably derived from apolynucleotide handling enzyme. The one or more molecular brakes may bederived from any of the polynucleotide handling enzymes discussed above.Modified versions of Phi29 polymerase (SEQ ID NO: 396) which act asmolecular brakes are disclosed in U.S. Pat. No. 5,576,204. The one ormore molecular brakes are preferably derived from a helicase.

Any number of molecular brakes derived from a helicase may be used. Forinstance, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases may be used asmolecular brakes. If two or more helicases are be used as molecularbrakes, the two or more helicases are typically the same helicase. Thetwo or more helicases may be different helicases.

The two or more helicases may be any combination of the helicasesmentioned above. The two or more helicases may be two or more Ddahelicases. The two or more helicases may be one or more Dda helicasesand one or more TrwC helicases. The two or more helicases may bedifferent variants of the same helicase.

The two or more helicases are preferably attached to one another. Thetwo or more helicases are more preferably covalently attached to oneanother. The helicases may be attached in any order and using anymethod. The one or more molecular brakes derived from helicases arepreferably modified to reduce the size of an opening in thepolynucleotide binding domain through which in at least oneconformational state the polynucleotide can unbind from the helicase.This is disclosed in WO 2014/013260.

Preferred helicase constructs for use in the invention are described inInternational Application Nos. PCT/GB2013/051925 (published as WO2014/013260); PCT/GB2013/051924 (published as WO 2014/013259);PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736.

If the one or more helicases are used in the active mode (i.e. when theone or more helicases are provided with all the necessary components tofacilitate movement, e.g. ATP and Mg²⁺), the one or more molecularbrakes are preferably (a) used in an inactive mode (i.e. are used in theabsence of the necessary components to facilitate movement or areincapable of active movement), (b) used in an active mode where the oneor more molecular brakes move in the opposite direction to the one ormore helicases or (c) used in an active mode where the one or moremolecular brakes move in the same direction as the one or more helicasesand more slowly than the one or more helicases.

If the one or more helicases are used in the inactive mode (i.e. whenthe one or more helicases are not provided with all the necessarycomponents to facilitate movement, e.g. ATP and Mg²⁺ or are incapable ofactive movement), the one or more molecular brakes are preferably (a)used in an inactive mode (i.e. are used in the absence of the necessarycomponents to facilitate movement or are incapable of active movement)or (b) used in an active mode where the one or more molecular brakesmove along the polynucleotide in the same direction as thepolynucleotide through the pore.

The one or more helicases and one or more molecular brakes may beattached to the polynucleotide at any positions so that they are broughttogether and both control the movement of the polynucleotide through thepore. The one or more helicases and one or more molecular brakes are atleast one nucleotide apart, such as at least 5, at least 10, at least50, at least 100, at least 500, at least 1000, at least 5000, at least10,000, at least 50,000 nucleotides or more apart. If the methodconcerns characterising a double stranded polynucleotide provided with aY adaptor at one end and a hairpin loop adaptor at the other end, theone or more helicases are preferably attached to the Y adaptor and theone or more molecular brakes are preferably attached to the hairpin loopadaptor. In this embodiment, the one or more molecular brakes arepreferably one or more helicases that are modified such that they bindthe polynucleotide but do not function as a helicase. The one or morehelicases attached to the Y adaptor are preferably stalled at a spaceras discussed in more detail below. The one or more molecular brakesattach to the hairpin loop adaptor are preferably not stalled at aspacer. The one or more helicases and the one or more molecular brakesare preferably brought together when the one or more helicases reach thehairpin loop. The one or more helicases may be attached to the Y adaptorbefore the Y adaptor is attached to the polynucleotide or after the Yadaptor is attached to the polynucleotide. The one or more molecularbrakes may be attached to the hairpin loop adaptor before the hairpinloop adaptor is attached to the polynucleotide or after the hairpin loopadaptor is attached to the polynucleotide.

The one or more helicases and the one or more molecular brakes arepreferably not attached to one another. The one or more helicases andthe one or more molecular brakes are more preferably not covalentlyattached to one another. The one or more helicases and the one or moremolecular brakes are preferably not attached as described inInternational Application Nos. PCT/GB2013/051925 (published as WO2014/013260); PCT/GB2013/051924 (published as WO 2014/013259);PCT/GB2013/051928 (published as WO 2014/013262) and PCT/GB2014/052736.

Spacers

The one or more helicases may be stalled at one or more spacers asdiscussed in International Application No. PCT/GB2014/050175. Anyconfiguration of one or more helicases and one or more spacers disclosedin the International Application may be used in this invention.

When a part of the polynucleotide enters the pore and moves through thepore along the field resulting from the applied potential, the one ormore helicases are moved past the spacer by the pore as thepolynucleotide moves through the pore. This is because thepolynucleotide (including the one or more spacers) moves through thepore and the one or more helicases remain on top of the pore.

The one or more spacers are preferably part of the polynucleotide, forinstance they interrupt(s) the polynucleotide sequence. The one or morespacers are preferably not part of one or more blocking molecules, suchas speed bumps, hybridised to the polynucleotide.

There may be any number of spacers in the polynucleotide, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more spacers. There are preferably two, fouror six spacers in the polynucleotide. There may be one or more spacersin different regions of the polynucleotide, such as one or more spacersin the Y adaptor and/or hairpin loop adaptor.

The one or more spacers each provides an energy barrier which the one ormore helicases cannot overcome even in the active mode. The one or morespacers may stall the one or more helicases by reducing the traction ofthe helicase (for instance by removing the bases from the nucleotides inthe polynucleotide) or physically blocking movement of the one or morehelicases (for instance using a bulky chemical group).

The one or more spacers may comprise any molecule or combination ofmolecules that stalls the one or more helicases. The one or more spacersmay comprise any molecule or combination of molecules that prevents theone or more helicases from moving along the polynucleotide. It isstraightforward to determine whether or not the one or more helicasesare staled at one or more spacers in the absence of a transmembrane poreand an applied potential. For instance, the ability of a helicase tomove past a spacer and displace a complementary strand of DNA can bemeasured by PAGE.

The one or more spacers typically comprise a linear molecule, such as apolymer. The one or more spacers typically have a different structurefrom the polynucleotide. For instance, if the polynucleotide is DNA, theone or more spacers are typically not DNA. In particular, if thepolynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),the one or more spacers preferably comprise peptide nucleic acid (PNA),glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleicacid (LNA) or a synthetic polymer with nucleotide side chains. The oneor more spacers may comprise one or more nucleotides in the oppositedirection from the polynucleotide. For instance, the one or more spacersmay comprise one or more nucleotides in the 3′ to 5′ direction when thepolynucleotide is in the 5′ to 3′ direction. The nucleotides may be anyof those discussed above.

The one or more spacers preferably comprises one or more nitroindoles,such as one or more 5-nitroindoles, one or more inosines, one or moreacridines, one or more 2-aminopurines, one or more 2-6-diaminopurines,one or more 5-bromo-deoxyuridines, one or more inverted thymidines(inverted dTs), one or more inverted dideoxy-thymidines (ddTs), one ormore dideoxy-cytidines (ddCs), one or more 5-methylcytidines, one ormore 5-hydroxymethylcytidines, one or more 2′-O-Methyl RNA bases, one ormore Iso-deoxycytidines (Iso-dCs), one or more Iso-deoxyguanosines(Iso-dGs), one or more iSpC3 groups (i.e. nucleotides which lack sugarand a base), one or more photo-cleavable (PC) groups, one or morehexanediol groups, one or more spacer 9 (iSp9) groups, one or morespacer 18 (iSp18) groups, a polymer or one or more thiol connections.The one or more spacers may comprise any combination of these groups.Many of these groups are commercially available from IDT® (IntegratedDNA Technologies®).

The one or more spacers may contain any number of these groups. Forinstance, for 2-aminopurines, 2-6-diaminopurines, 5-bromo-deoxyuridines,inverted dTs, ddTs, ddCs, 5-methylcytidines, 5-hydroxymethylcytidines,2′-O-Methyl RNA bases, Iso-dCs, Iso-dGs, iSpC3 groups, PC groups,hexanediol groups and thiol connections, the one or more spacerspreferably comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. The oneor more spacers preferably comprise 2, 3, 4, 5, 6, 7, 8 or more iSp9groups. The one or more spacers preferably comprise 2, 3, 4, 5 or 6 ormore iSp18 groups. The most preferred spacer is four iSp18 groups.

The polymer is preferably a polypeptide or a polyethylene glycol (PEG).The polypeptide preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12or more amino acids. The PEG preferably comprises 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12 or more monomer units.

The one or more spacers preferably comprise one or more abasicnucleotides (i.e. nucleotides lacking a nucleobase), such as 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides. The nucleobase can bereplaced by —H (idSp) or —OH in the abasic nucleotide. Abasic spacerscan be inserted into polynucleotides by removing the nucleobases fromone or more adjacent nucleotides. For instance, polynucleotides may bemodified to include 3-methyladenine, 7-methylguanine, 1,N6-ethenoadenineinosine or hypoxanthine and the nucleobases may be removed from thesenucleotides using Human Alkyladenine DNA Glycosylase (hAAG).Alternatively, polynucleotides may be modified to include uracil and thenucleobases removed with Uracil-DNA Glycosylase (UDG). In oneembodiment, the one or more spacers do not comprise any abasicnucleotides.

The one or more helicases may be stalled by (i.e. before) or on eachlinear molecule spacers. If linear molecule spacers are used, thepolynucleotide is preferably provided with a double stranded region ofpolynucleotide adjacent to the end of each spacer past which the one ormore helicases are to be moved. The double stranded region typicallyhelps to stall the one or more helicases on the adjacent spacer. Thepresence of the double stranded region(s) is particularly preferred ifthe method is carried out at a salt concentration of about 100 mM orlower. Each double stranded region is typically at least 10, such as atleast 12, nucleotides in length. If the polynucleotide used in theinvention is single stranded, a double stranded region may be formed byhybridising a shorter polynucleotide to a region adjacent to a spacer.The shorter polynucleotide is typically formed from the same nucleotidesas the polynucleotide, but may be formed from different nucleotides. Forinstance, the shorter polynucleotide may be formed from LNA.

If linear molecule spacers are used, the polynucleotide is preferablyprovided with a blocking molecule at the end of each spacer opposite tothe end past which the one or more helicases are to be moved. This canhelp to ensure that the one or more helicases remain stalled on eachspacer. It may also help retain the one or more helicases on thepolynucleotide in the case that it/they diffuse(s) off in solution. Theblocking molecule may be any of the chemical groups discussed belowwhich physically cause the one or more helicases to stall. The blockingmolecule may be a double stranded region of polynucleotide.

The one or more spacers preferably comprise one or more chemical groupswhich physically cause the one or more helicases to stall. The one ormore chemical groups are preferably one or more pendant chemical groups.The one or more chemical groups may be attached to one or morenucleobases in the polynucleotide. The one or more chemical groups maybe attached to the polynucleotide backbone. Any number of these chemicalgroups may be present, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 ormore. Suitable groups include, but are not limited to, fluorophores,streptavidin and/or biotin, cholesterol, methylene blue, dinitrophenols(DNPs), digoxigenin and/or anti-digoxigenin and dibenzylcyclooctynegroups.

Different spacers in the polynucleotide may comprise different stallingmolecules. For instance, one spacer may comprise one of the linearmolecules discussed above and another spacer may comprise one or morechemical groups which physically cause the one or more helicases tostall. A spacer may comprise any of the linear molecules discussed aboveand one or more chemical groups which physically cause the one or morehelicases to stall, such as one or more abasics and a fluorophore.

Suitable spacers can be designed depending on the type of polynucleotideand the conditions under which the method of the invention is carriedout. Most helicases bind and move along DNA and so may be stalled usinganything that is not DNA. Suitable molecules are discussed above.

The method of the invention is preferably carried out in the presence offree nucleotides and/or the presence of a helicase cofactor. This isdiscussed in more detail below. In the absence of the transmembrane poreand an applied potential, the one or more spacers are preferably capableof stalling the one or more helicases in the presence of freenucleotides and/or the presence of a helicase cofactor.

If the method of the invention is carried out in the presence of freenucleotides and a helicase cofactor as discussed below (such that theone of more helicases are in the active mode), one or more longerspacers are typically used to ensure that the one or more helicases arestalled on the polynucleotide before they are contacted with thetransmembrane pore and a potential is applied. One or more shorterspacers may be used in the absence of free nucleotides and a helicasecofactor (such that the one or more helicases are in the inactive mode).

The salt concentration also affects the ability of the one or morespacers to stall the one or more helicases. In the absence of thetransmembrane pore and an applied potential, the one or more spacers arepreferably capable of stalling the one or more helicases at a saltconcentration of about 100 mM or lower. The higher the saltconcentration used in the method of the invention, the shorter the oneor more spacers that are typically used and vice versa.

Preferred combinations of features are shown in Table 3 below.

TABLE 3 Spacer length Spacer (i.e. Poly- compo- number Free Helicasenucleotide sition* of*) Salt [ ] nucleotides? cofactor? DNA iSpC3 4 1MYes Yes DNA iSp18 4   100-1000 mM Yes Yes DNA iSp18 6 <100-1000 mM YesYes DNA iSp18 2 1 M Yes Yes DNA iSpC3 12 <100-1000 mM Yes Yes DNA iSpC320 <100-1000 mM Yes Yes DNA iSp9 6   100-1000 mM Yes Yes DNA idSp 4 1MYes Yes

The method may concern moving two or more helicases past a spacer. Insuch instances, the length of the spacer is typically increased toprevent the trailing helicase from pushing the leading helicase past thespacer in the absence of the pore and applied potential. If the methodconcerns moving two or more helicases past one or more spacers, thespacer lengths discussed above may be increased at least 1.5 fold, such2 fold, 2.5 fold or 3 fold. For instance, if the method concerns movingtwo or more helicases past one or more spacers, the spacer lengths inthe third column of Table 3 above may be increased 1.5 fold, 2 fold, 2.5fold or 3 fold.

Membrane

The pore of the invention may be present in a membrane. In the methodsof the invention, the polynucleotide is typically contacted with theCsgG pore or mutant thereof, such as a pore of the invention, in amembrane. Any membrane may be used in accordance with the invention.Suitable membranes are well-known in the art. The membrane is preferablyan amphiphilic layer. An amphiphilic layer is a layer formed fromamphiphilic molecules, such as phospholipids, which have bothhydrophilic and lipophilic properties. The amphiphilic molecules may besynthetic or naturally occurring. Non-naturally occurring amphiphilesand amphiphiles which form a monolayer are known in the art and include,for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009,25, 10447-10450). Block copolymers are polymeric materials in which twoor more monomer sub-units that are polymerized together to create asingle polymer chain. Block copolymers typically have properties thatare contributed by each monomer sub-unit. However, a block copolymer mayhave unique properties that polymers formed from the individualsub-units do not possess. Block copolymers can be engineered such thatone of the monomer sub-units is hydrophobic (i.e. lipophilic), whilstthe other sub-unit(s) are hydrophilic whilst in aqueous media. In thiscase, the block copolymer may possess amphiphilic properties and mayform a structure that mimics a biological membrane. The block copolymermay be a diblock (consisting of two monomer sub-units), but may also beconstructed from more than two monomer sub-units to form more complexarrangements that behave as amphiphiles. The copolymer may be atriblock, tetrablock or pentablock copolymer. The membrane is preferablya triblock copolymer membrane.

Archaebacterial bipolar tetraether lipids are naturally occurring lipidsthat are constructed such that the lipid forms a monolayer membrane.These lipids are generally found in extremophiles that survive in harshbiological environments, thermophiles, halophiles and acidophiles. Theirstability is believed to derive from the fused nature of the finalbilayer. It is straightforward to construct block copolymer materialsthat mimic these biological entities by creating a triblock polymer thathas the general motif hydrophilic-hydrophobic-hydrophilic. This materialmay form monomeric membranes that behave similarly to lipid bilayers andencompass a range of phase behaviours from vesicles through to laminarmembranes. Membranes formed from these triblock copolymers hold severaladvantages over biological lipid membranes. Because the triblockcopolymer is synthesised, the exact construction can be carefullycontrolled to provide the correct chain lengths and properties requiredto form membranes and to interact with pores and other proteins.

Block copolymers may also be constructed from sub-units that are notclassed as lipid sub-materials; for example a hydrophobic polymer may bemade from siloxane or other non-hydrocarbon based monomers. Thehydrophilic sub-section of block copolymer can also possess low proteinbinding properties, which allows the creation of a membrane that ishighly resistant when exposed to raw biological samples. This head groupunit may also be derived from non-classical lipid head-groups.

Triblock copolymer membranes also have increased mechanical andenvironmental stability compared with biological lipid membranes, forexample a much higher operational temperature or pH range. The syntheticnature of the block copolymers provides a platform to customise polymerbased membranes for a wide range of applications.

The membrane is most preferably one of the membranes disclosed inInternational Application No. PCT/GB2013/052766 or PCT/GB2013/052767.

The amphiphilic molecules may be chemically-modified or functionalisedto facilitate coupling of the polynucleotide.

The amphiphilic layer may be a monolayer or a bilayer. The amphiphiliclayer is typically planar. The amphiphilic layer may be curved. Theamphiphilic layer may be supported.

Amphiphilic membranes are typically naturally mobile, essentially actingas two dimensional fluids with lipid diffusion rates of approximately10⁻⁸ cm s-1. This means that the pore and coupled polynucleotide cantypically move within an amphiphilic membrane.

The membrane may be a lipid bilayer. Lipid bilayers are models of cellmembranes and serve as excellent platforms for a range of experimentalstudies. For example, lipid bilayers can be used for in vitroinvestigation of membrane proteins by single-channel recording.Alternatively, lipid bilayers can be used as biosensors to detect thepresence of a range of substances. The lipid bilayer may be any lipidbilayer. Suitable lipid bilayers include, but are not limited to, aplanar lipid bilayer, a supported bilayer or a liposome. The lipidbilayer is preferably a planar lipid bilayer. Suitable lipid bilayersare disclosed in International Application No. PCT/GB08/000563(published as WO 2008/102121), International Application No.PCT/GB08/004127 (published as WO 2009/077734) and InternationalApplication No. PCT/GB2006/001057 (published as WO 2006/100484).

Methods for forming lipid bilayers are known in the art. Lipid bilayersare commonly formed by the method of Montal and Mueller (Proc. Natl.Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer iscarried on aqueous solution/air interface past either side of anaperture which is perpendicular to that interface. The lipid is normallyadded to the surface of an aqueous electrolyte solution by firstdissolving it in an organic solvent and then allowing a drop of thesolvent to evaporate on the surface of the aqueous solution on eitherside of the aperture. Once the organic solvent has evaporated, thesolution/air interfaces on either side of the aperture are physicallymoved up and down past the aperture until a bilayer is formed. Planarlipid bilayers may be formed across an aperture in a membrane or acrossan opening into a recess.

The method of Montal & Mueller is popular because it is a cost-effectiveand relatively straightforward method of forming good quality lipidbilayers that are suitable for protein pore insertion. Other commonmethods of bilayer formation include tip-dipping, painting bilayers andpatch-clamping of liposome bilayers.

Tip-dipping bilayer formation entails touching the aperture surface (forexample, a pipette tip) onto the surface of a test solution that iscarrying a monolayer of lipid. Again, the lipid monolayer is firstgenerated at the solution/air interface by allowing a drop of lipiddissolved in organic solvent to evaporate at the solution surface. Thebilayer is then formed by the Langmuir-Schaefer process and requiresmechanical automation to move the aperture relative to the solutionsurface.

For painted bilayers, a drop of lipid dissolved in organic solvent isapplied directly to the aperture, which is submerged in an aqueous testsolution. The lipid solution is spread thinly over the aperture using apaintbrush or an equivalent Thinning of the solvent results in formationof a lipid bilayer. However, complete removal of the solvent from thebilayer is difficult and consequently the bilayer formed by this methodis less stable and more prone to noise during electrochemicalmeasurement.

Patch-clamping is commonly used in the study of biological cellmembranes. The cell membrane is clamped to the end of a pipette bysuction and a patch of the membrane becomes attached over the aperture.The method has been adapted for producing lipid bilayers by clampingliposomes which then burst to leave a lipid bilayer sealing over theaperture of the pipette. The method requires stable, giant andunilamellar liposomes and the fabrication of small apertures inmaterials having a glass surface.

Liposomes can be formed by sonication, extrusion or the Mozafari method(Colas et al. (2007) Micron 38:841-847).

In a preferred embodiment, the lipid bilayer is formed as described inInternational Application No. PCT/GB08/004127 (published as WO2009/077734). Advantageously in this method, the lipid bilayer is formedfrom dried lipids. In a most preferred embodiment, the lipid bilayer isformed across an opening as described in WO2009/077734(PCT/GB08/004127).

A lipid bilayer is formed from two opposing layers of lipids. The twolayers of lipids are arranged such that their hydrophobic tail groupsface towards each other to form a hydrophobic interior. The hydrophilichead groups of the lipids face outwards towards the aqueous environmenton each side of the bilayer. The bilayer may be present in a number oflipid phases including, but not limited to, the liquid disordered phase(fluid lamellar), liquid ordered phase, solid ordered phase (lamellargel phase, Interdigitated gel phase) and planar bilayer crystals(lamellar sub-gel phase, lamellar crystalline phase).

Any lipid composition that forms a lipid bilayer may be used. The lipidcomposition is chosen such that a lipid bilayer having the requiredproperties, such surface charge, ability to support membrane proteins,packing density or mechanical properties, is formed. The lipidcomposition can comprise one or more different lipids. For instance, thelipid composition can contain up to 100 lipids. The lipid compositionpreferably contains 1 to 10 lipids. The lipid composition may comprisenaturally-occurring lipids and/or artificial lipids.

The lipids typically comprise a head group, an interfacial moiety andtwo hydrophobic tail groups which may be the same or different. Suitablehead groups include, but are not limited to, neutral head groups, suchas diacylglycerides (DG) and ceramides (CM); zwitterionic head groups,such as phosphatidylcholine (PC), phosphatidylethanolemine (PE) andsphingomyelin (SM); negatively charged head groups, such asphosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol(PI), phosphatic acid (PA) and cardiolipin (CA); and positively chargedheadgroups, such as trimethylammonium-Propane (TAP). Suitableinterfacial moieties include, but are not limited to,naturally-occurring interfacial moieties, such as glycerol-based orceramide-based moieties. Suitable hydrophobic tail groups include, butare not limited to, saturated hydrocarbon chains, such as lauric acid(n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmiticacid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic(n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid(cis-9-Octadecanoic); and branched hydrocarbon chains, such asphytanoyl. The length of the chain and the position and number of thedouble bonds in the unsaturated hydrocarbon chains can vary. The lengthof the chains and the position and number of the branches, such asmethyl groups, in the branched hydrocarbon chains can vary. Thehydrophobic tail groups can be linked to the interfacial moiety as anether or an ester. The lipids may be mycolic acid.

The lipids can also be chemically-modified. The head group or the tallgroup of the lipids may be chemically-modified. Suitable lipids whosehead groups have been chemically-modified include, but are not limitedto, PEG-modified lipids, such as1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethyleneglycol)-2000]; functionalised PEG Lipids, such as1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(PolyethyleneGlycol)2000]; and lipids modified for conjugation, such as1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolemine-N-(succinyl) and1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl). Suitablelipids whose tail groups have been chemically-modified include, but arenot limited to, polymerisable lipids, such as1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinatedlipids, such as1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine;deuterated lipids, such as1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linkedlipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. Thelipids may be chemically-modified or functionalised to facilitatecoupling of the polynucleotide.

The amphiphilic layer, for example the lipid composition, typicallycomprises one or more additives that will affect the properties of thelayer. Suitable additives include, but are not limited to, fatty acids,such as palmitic acid, myristic acid and oleic acid; fatty alcohols,such as palmitic alcohol, myristic alcohol and oleic alcohol; sterols,such as cholesterol, ergosterol, lanosterol, sitosterol andstigmasterol; lysophospholipids, such as1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; and ceramides.

In another preferred embodiment, the membrane comprises a solid statelayer. Solid state layers can be formed from both organic and inorganicmaterials including, but not limited to, microelectronic materials,insulating materials such as Si₃N₄, A1₂O₃, and SiO, organic andinorganic polymers such as polyamide, plastics such as Teflon® orelastomers such as two-component addition-cure silicone rubber, andglasses. The solid state layer may be formed from graphene. Suitablegraphene layers are disclosed in International Application No.PCT/US2008/010637 (published as WO 2009/035647). If the membranecomprises a solid state layer, the pore is typically present in anamphiphilic membrane or layer contained within the solid state layer,for instance within a hole, well, gap, channel, trench or slit withinthe solid state layer. The skilled person can prepare suitable solidstate/amphiphilic hybrid systems. Suitable systems are disclosed in WO2009/020682 and WO 2012/005857. Any of the amphiphilic membranes orlayers discussed above may be used.

The method is typically carried out using (i) an artificial amphiphiliclayer comprising a pore, (ii) an isolated, naturally-occurring lipidbilayer comprising a pore, or (iii) a cell having a pore insertedtherein. The method is typically carried out using an artificialamphiphilic layer, such as an artificial triblock copolymer layer. Thelayer may comprise other transmembrane and/or intramembrane proteins aswell as other molecules in addition to the pore. Suitable apparatus andconditions are discussed below. The method of the invention is typicallycarried out in vitro.

Coupling

The polynucleotide is preferably coupled to the membrane comprising thepore. The method may comprise coupling the polynucleotide to themembrane comprising the pore. The polynucleotide is preferably coupledto the membrane using one or more anchors. The polynucleotide may becoupled to the membrane using any known method.

Each anchor comprises a group which couples (or binds) to thepolynucleotide and a group which couples (or binds) to the membrane.Each anchor may covalently couple (or bind) to the polynucleotide and/orthe membrane. If a Y adaptor and/or a hairpin loop adaptors are used,the polynucleotide is preferably coupled to the membrane using theadaptor(s).

The polynucleotide may be coupled to the membrane using any number ofanchors, such as 2, 3, 4 or more anchors. For instance, a polynucleotidemay be coupled to the membrane using two anchors each of whichseparately couples (or binds) to both the polynucleotide and membrane.

The one or more anchors may comprise the one or more helicases and/orthe one or more molecular brakes.

If the membrane is an amphiphilic layer, such as a copolymer membrane ora lipid bilayer, the one or more anchors preferably comprise apolypeptide anchor present in the membrane and/or a hydrophobic anchorpresent in the membrane. The hydrophobic anchor is preferably a lipid,fatty acid, sterol, carbon nanotube, polypeptide, protein or amino acid,for example cholesterol, palmitate or tocopherol. In preferredembodiments, the one or more anchors are not the pore.

The components of the membrane, such as the amphiphilic molecules,copolymer or lipids, may be chemically-modified or functionalised toform the one or more anchors. Examples of suitable chemicalmodifications and suitable ways of functionalising the components of themembrane are discussed in more detail below. Any proportion of themembrane components may be functionalised, for example at least 0.01%,at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or100%.

The polynucleotide may be coupled directly to the membrane. The one ormore anchors used to couple the polynucleotide to the membranepreferably comprise a linker. The one or more anchors may comprise oneor more, such as 2, 3, 4 or more, linkers. One linker may be used couplemore than one, such as 2, 3, 4 or more, polynucleotides to the membrane.

Preferred linkers include, but are not limited to, polymers, such aspolynucleotides, polyethylene glycols (PEGs), polysaccharides andpolypeptides. These linkers may be linear, branched or circular. Forinstance, the linker may be a circular polynucleotide. Thepolynucleotide may hybridise to a complementary sequence on the circularpolynucleotide linker.

The one or more anchors or one or more linkers may comprise a componentthat can be cut to broken down, such as a restriction site or aphotolabile group.

Functionalised linkers and the ways in which they can couple moleculesare known in the art. For instance, linkers functionalised withmaleimide groups will react with and attach to cysteine residues inproteins. In the context of this invention, the protein may be presentin the membrane or may be used to couple (or bind) to thepolynucleotide. This is discussed in more detail below.

Crosslinkage of polynucleotides can be avoided using a “lock and key”arrangement. Only one end of each linker may react together to form alonger linker and the other ends of the linker each react with thepolynucleotide or membrane respectively. Such linkers are described inInternational Application No. PCT/GB10/000132 (published as WO2010/086602).

The use of a linker is preferred in the sequencing embodiments discussedbelow. If a polynucleotide is permanently coupled directly to themembrane in the sense that it does not uncouple when interacting withthe pore (i.e. does not uncouple in step (b) or (e)), then some sequencedata will be lost as the sequencing run cannot continue to the end ofthe polynucleotide due to the distance between the membrane and thepore. If a linker is used, then the polynucleotide can be processed tocompletion.

The coupling may be permanent or stable. In other words, the couplingmay be such that the polynucleotide remains coupled to the membrane wheninteracting with the pore.

The coupling may be transient. In other words, the coupling may be suchthat the polynucleotide may decouple from the membrane when interactingwith the pore.

For certain applications, such as aptamer detection, the transientnature of the coupling is preferred. If a permanent or stable linker isattached directly to either the 5′ or 3′ end of a polynucleotide and thelinker is shorter than the distance between the membrane and thetransmembrane pore's channel, then some sequence data will be lost asthe sequencing run cannot continue to the end of the polynucleotide. Ifthe coupling is transient, then when the coupled end randomly becomesfree of the membrane, then the polynucleotide can be processed tocompletion. Chemical groups that form permanent/stable or transientlinks are discussed in more detail below. The polynucleotide may betransiently coupled to an amphiphilic layer or triblock copolymermembrane using cholesterol or a fatty acyl chain. Any fatty acyl chainhaving a length of from 6 to 30 carbon atom, such as hexadecanoic acid,may be used.

In preferred embodiments, a polynucleotide, such as a nucleic acid, iscoupled to an amphiphilic layer such as a triblock copolymer membrane orlipid bilayer. Coupling of nucleic acids to synthetic lipid bilayers hasbeen carried out previously with various different tethering strategies.These are summarised in Table 4 below.

TABLE 4 Anchor comprising Type of coupling Reference Thiol StableYoshina-Ishii, C. and S. G. Boxer (2003). “Arrays of mobile tetheredvesicles on supported lipid bilayers.” J Am Chem Soc 125(13): 3696-7.Biotin Stable Nikolov, V., R. Lipowsky, et al. (2007). “Behavior ofgiant vesicles with anchored DNA molecules.” Biophys J 92(12): 4356-68Cholesterol Transient Pfeiffer, I. and F. Hook (2004). “Bivalentcholesterol-based coupling of oligonucletides to lipid membraneassemblies.” J Am Chem Soc 126(33): 10224-5 Surfactant (e.g. Lipid,Stable van Lengerich, B., R. J. Rawle, et al. “Covalent Palmitate, etc)attachment of lipid vesicles to a fluid-supported bilayer allowsobservation of DNA-mediated vesicle interactions.” Langmuir 26(11):8666-72

Synthetic polynucleotides and/or linkers may be functionalised using amodified phosphoramidite in the synthesis reaction, which is easilycompatible for the direct addition of suitable anchoring groups, such ascholesterol, tocopherol, palmitate, thiol, lipid and biotin groups.These different attachment chemistries give a suite of options forattachment to polynucleotides. Each different modification group couplesthe polynucleotide in a slightly different way and coupling is notalways permanent so giving different dwell times for the polynucleotideto the membrane. The advantages of transient coupling are discussedabove.

Coupling of polynucleotides to a linker or to a functionalised membranecan also be achieved by a number of other means provided that acomplementary reactive group or an anchoring group can be added to thepolynucleotide. The addition of reactive groups to either end of apolynucleotide has been reported previously. A thiol group can be addedto the 5′ of ssDNA or dsDNA using T4 polynucleotide kinase and ATPγS(Grant, G. P. and P. Z. Qin (2007). “A facile method for attachingnitroxide spin labels at the 5′ terminus of nucleic acids.” NucleicAcids Res 35(10): e77). An azide group can be added to the 5′-phosphateof ssDNA or dsDNA using T4 polynucleotide kinase andγ-[2-Azidoethyl]-ATP or γ-[6-Azidohexyl]-ATP. Using thiol or Clickchemistry a tether, containing either a thiol, iodoacetamide OPSS ormaleimide group (reactive to thiols) or a DIBO (dibenzocyclooxtyne) oralkyne group (reactive to azides), can be covalently attached to thepolynucleotide. A more diverse selection of chemical groups, such asbiotin, thiols and fluorophores, can be added using terminal transferaseto incorporate modified oligonucleotides to the 3′ of ssDNA (Kumar, A.,P. Tchen, et al. (1988). “Nonradioactive labeling of syntheticoligonucleotide probes with terminal deoxynucleotidyl transferase.” AnalBiochem 169(2): 376-82). Streptavidin/biotin and/orstreptavidin/desthiobiotin coupling may be used for any otherpolynucleotide. The Examples below describes how a polynucleotide can becoupled to a membrane using streptavidin/biotin andstreptavidin/desthiobiotin. It may also be possible that anchors may bedirectly added to polynucleotides using terminal transferase withsuitably modified nucleotides (e.g. cholesterol or palmitate).

The one or more anchors preferably couple the polynucleotide to themembrane via hybridisation. Hybridisation in the one or more anchorsallows coupling in a transient manner as discussed above. Thehybridisation may be present in any part of the one or more anchors,such as between the one or more anchors and the polynucleotide, withinthe one or more anchors or between the one or more anchors and themembrane. For instance, a linker may comprise two or morepolynucleotides, such as 3, 4 or 5 polynucleotides, hybridised together.The one or more anchors may hybridise to the polynucleotide. The one ormore anchors may hybridise directly to the polynucleotide or directly toa Y adaptor and/or leader sequence attached to the polynucleotide ordirectly to a hairpin loop adaptor attached to the polynucleotide (asdiscussed below). Alternatively, the one or more anchors may behybridised to one or more, such as 2 or 3, intermediate polynucleotides(or “splints”) which are hybridised to the polynucleotide, to a Yadaptor and/or leader sequence attached to the polynucleotide or to ahairpin loop adaptor attached to the polynucleotide (as discussedbelow).

The one or more anchors may comprise a single stranded or doublestranded polynucleotide. One part of the anchor may be ligated to asingle stranded or double stranded polynucleotide. Ligation of shortpieces of ssDNA have been reported using T4 RNA ligase I (Troutt, A. B.,M. G. McHeyzer-Williams, et al. (1992). “Ligation-anchored PCR: a simpleamplification technique with single-sided specificity.” Proc Natl AcadSci USA 89(20): 9823-5). Alternatively, either a single stranded ordouble stranded polynucleotide can be ligated to a double strandedpolynucleotide and then the two strands separated by thermal or chemicaldenaturation. To a double stranded polynucleotide, it is possible to addeither a piece of single stranded polynucleotide to one or both of theends of the duplex, or a double stranded polynucleotide to one or bothends. For addition of single stranded polynucleotides to the a doublestranded polynucleotide, this can be achieved using T4 RNA ligase I asfor ligation to other regions of single stranded polynucleotides. Foraddition of double stranded polynucleotides to a double strandedpolynucleotide then ligation can be “blunt-ended”, with complementary 3′dA/dT tails on the polynucleotide and added polynucleotide respectively(as is routinely done for many sample prep applications to preventconcatemer or dimer formation) or using “sticky-ends” generated byrestriction digestion of the polynucleotide and ligation of compatibleadapters. Then, when the duplex is melted, each single strand will haveeither a 5′ or 3′ modification if a single stranded polynucleotide wasused for ligation or a modification at the 5′ end, the 3′ end or both ifa double stranded polynucleotide was used for ligation.

If the polynucleotide is a synthetic strand, the one or more anchors canbe incorporated during the chemical synthesis of the polynucleotide. Forinstance, the polynucleotide can be synthesised using a primer having areactive group attached to it.

Adenylated polynucleotides are intermediates in ligation reactions,where an adenosine-monophosphate is attached to the 5′-phosphate of thepolynucleotide. Various kits are available for generation of thisintermediate, such as the 5′ DNA Adenylation Kit from NEB. Bysubstituting ATP in the reaction for a modified nucleotide triphosphate,then addition of reactive groups (such as thiols, amines, biotin,azides, etc) to the 5′ of a polynucleotide can be possible. It may alsobe possible that anchors could be directly added to polynucleotidesusing a 5′ DNA adenylation kit with suitably modified nucleotides (e.g.cholesterol or palmitate).

A common technique for the amplification of sections of genomic DNA isusing polymerase chain reaction (PCR). Here, using two syntheticoligonucleotide primers, a number of copies of the same section of DNAcan be generated, where for each copy the 5′ of each strand in theduplex will be a synthetic polynucleotide. Single or multiplenucleotides can be added to 3′ end of single or double stranded DNA byemploying a polymerase. Examples of polymerases which could be usedinclude, but are not limited to, Terminal Transferase, Klenow and E.coli Poly(A) polymerase). By substituting ATP in the reaction for amodified nucleotide triphosphate then anchors, such as a cholesterol,thiol, amine, azide, biotin or lipid, can be incorporated into doublestranded polynucleotides. Therefore, each copy of the amplifiedpolynucleotide will contain an anchor.

Ideally, the polynucleotide is coupled to the membrane without having tofunctionalise the polynucleotide. This can be achieved by coupling theone or more anchors, such as a polynucleotide binding protein or achemical group, to the membrane and allowing the one or more anchors tointeract with the polynucleotide or by functionalising the membrane. Theone or more anchors may be coupled to the membrane by any of the methodsdescribed herein. In particular, the one or more anchors may compriseone or more linkers, such as maleimide functionalised linkers.

In this embodiment, the polynucleotide is typically RNA, DNA, PNA, TNAor LNA and may be double or single stranded. This embodiment isparticularly suited to genomic DNA polynucleotides.

The one or more anchors can comprise any group that couples to, binds toor interacts with single or double stranded polynucleotides, specificnucleotide sequences within the polynucleotide or patterns of modifiednucleotides within the polynucleotide, or any other ligand that ispresent on the polynucleotide.

Suitable binding proteins for use in anchors include, but are notlimited to, E. coli single stranded binding protein, P5 single strandedbinding protein, T4 gp32 single stranded binding protein, the TOPO VdsDNA binding region, human histone proteins, E. coli HU DNA bindingprotein and other archaeal, prokaryotic or eukaryotic single stranded ordouble stranded polynucleotide (or nucleic acid) binding proteins,including those listed below.

The specific nucleotide sequences could be sequences recognised bytranscription factors, ribosomes, endonucleases, topoisomerases orreplication initiation factors. The patterns of modified nucleotidescould be patterns of methylation or damage.

The one or more anchors can comprise any group which couples to, bindsto, intercalates with or interacts with a polynucleotide. The group mayintercalate or interact with the polynucleotide via electrostatic,hydrogen bonding or Van der Waals interactions. Such groups include alysine monomer, poly-lysine (which will interact with ssDNA or dsDNA),ethidium bromide (which will intercalate with dsDNA), universal bases oruniversal nucleotides (which can hybridise with any polynucleotide) andosmium complexes (which can react to methylated bases). A polynucleotidemay therefore be coupled to the membrane using one or more universalnucleotides attached to the membrane. Each universal nucleotide may becoupled to the membrane using one or more linkers. The universalnucleotide preferably comprises one of the following nucleobases:hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, formylindole,3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole,5-nitroindazole, 4-aminobenzimidazole or phenyl (C6-aromatic ring). Theuniversal nucleotide more preferably comprises one of the followingnucleosides: 2′-deoxyinosine, inosine, 7-deaza-2′-deoxyinosine,7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 2-O′-methylinosine,4-nitroindole 2′-deoxyribonucleoside, 4-nitroindole ribonucleoside,5-nitroindole 2′-deoxyribonucleoside, 5-nitroindole ribonucleoside,6-nitroindole 2′-deoxyribonucleoside, 6-nitroindole ribonucleoside,3-nitropyrrole 2′-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, anacyclic sugar analogue of hypoxanthine, nitroimidazole2′-deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole2-deoxyribonucleoside, 4-nitropyrazole ribonucleoside,4-nitrobenzimidazole 2′-deoxyribonucleoside, 4-nitrobenzimidazoleribonucleoside, 5-nitroindazole 2′-deoxyribonucleoside, 5-nitroindazoleribonucleoside, 4-aminobenzimidazole 2′-deoxyribonucleoside,4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside, phenylC-2′-deoxyribosyl nucleoside, 2′-deoxynebularine, 2′-deoxyisoguanosine,K-2′-deoxyribose, P-2′-deoxyribose and pyrrolidine. The universalnucleotide more preferably comprises 2′-deoxyinosine. The universalnucleotide is more preferably IMP or dIMP. The universal nucleotide ismost preferably dPMP (2′-Deoxy-P-nucleoside monophosphate) or dKMP(N6-methoxy-2, 6-diaminopurine monophosphate).

The one or more anchors may couple to (or bind to) the polynucleotidevia Hoogsteen hydrogen bonds (where two nucleobases are held together byhydrogen bonds) or reversed Hoogsteen hydrogen bonds (where onenucleobase is rotated through 180° with respect to the othernucleobase). For instance, the one or more anchors may comprise one ormore nucleotides, one or more oligonucleotides or one or morepolynucleotides which form Hoogsteen hydrogen bonds or reversedHoogsteen hydrogen bonds with the polynucleotide. These types ofhydrogen bonds allow a third polynucleotide strand to wind around adouble stranded helix and form a triplex. The one or more anchors maycouple to (or bind to) a double stranded polynucleotide by forming atriplex with the double stranded duplex.

In this embodiment at least 1%, at least 10%, at least 25%, at least 50%or 100% of the membrane components may be functionalised.

Where the one or more anchors comprise a protein, they may be able toanchor directly into the membrane without further functonalisation, forexample if it already has an external hydrophobic region which iscompatible with the membrane. Examples of such proteins include, but arenot limited to, transmembrane proteins, intramembrane proteins andmembrane proteins. Alternatively the protein may be expressed with agenetically fused hydrophobic region which is compatible with themembrane. Such hydrophobic protein regions are known in the art.

The one or more anchors are preferably mixed with the polynucleotidebefore contacting with the membrane, but the one or more anchors may becontacted with the membrane and subsequently contacted with thepolynucleotide.

In another aspect the polynucleotide may be functionalised, usingmethods described above, so that it can be recognised by a specificbinding group. Specifically the polynucleotide may be functionalisedwith a ligand such as biotin (for binding to streptavidin), amylose (forbinding to maltose binding protein or a fusion protein), Ni-NTA (forbinding to poly-histidine or poly-histidine tagged proteins) or apeptides (such as an antigen).

According to a preferred embodiment, the one or more anchors may be usedto couple a polynucleotide to the membrane when the polynucleotide isattached to a leader sequence which preferentially threads into thepore. Leader sequences are discussed in more detail below. Preferably,the polynucleotide is attached (such as ligated) to a leader sequencewhich preferentially threads into the pore. Such a leader sequence maycomprise a homopolymeric polynucleotide or an abasic region. The leadersequence is typically designed to hybridise to the one or more anchorseither directly or via one or more intermediate polynucleotides (orsplints). In such instances, the one or more anchors typically comprisea polynucleotide sequence which is complementary to a sequence in theleader sequence or a sequence in the one or more intermediatepolynucleotides (or splints). In such instances, the one or more splintstypically comprise a polynucleotide sequence which is complementary to asequence in the leader sequence.

An example of a molecule used in chemical attachment is EDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride). Reactivegroups can also be added to the 5′ of polynucleotides using commerciallyavailable kits (Thermo Pierce, Part No. 22980). Suitable methodsinclude, but are not limited to, transient affinity attachment usinghistidine residues and Ni-NTA, as well as more robust covalentattachment by reactive cysteines, lysines or non natural amino acids.

Double Stranded Polynucleotide

The polynucleotide may be double stranded. If the polynucleotide isdouble stranded, the method preferably further comprises before thecontacting step ligating a bridging moiety, such as a hairpin loop, toone end of the polynucleotide. The two strands of the polynucleotide maythen be separated as or before the polynucleotide is contacted with thepore in accordance with the invention. The two strands may be separatedas the polynucleotide movement through the pore is controlled by apolynucleotide binding protein, such as a helicase, or molecular brake.

Linking and interrogating both strands on a double stranded construct inthis way increases the efficiency and accuracy of characterisation.

The bridging moiety is capable of linking the two strands of the targetpolynucleotide. The bridging moiety typically covalently links the twostrands of the target polynucleotide. The bridging moiety can beanything that is capable of linking the two strands of the targetpolynucleotide, provided that the bridging moiety does not interferewith movement of the single stranded polynucleotide through thetransmembrane pore.

The bridging moiety may be linked to the target polynucleotide by anysuitable means known in the art. The bridging moiety may be synthesisedseparately and chemically attached or enzymatically ligated to thetarget polynucleotide. Alternatively, the bridging moiety may begenerated in the processing of the target polynucleotide.

The bridging moiety is linked to the target polynucleotide at or nearone end of the target polynucleotide. The bridging moiety is preferablylinked to the target polynucleotide within 10 nucleotides of the end ofthe target polynucleotide

Suitable bridging moieties include, but are not limited to a polymericlinker, a chemical linker, a polynucleotide or a polypeptide.Preferably, the bridging moiety comprises DNA, RNA, modified DNA (suchas abasic DNA), RNA, PNA, LNA or PEG. The bridging moiety is morepreferably DNA or RNA.

The bridging moiety is most preferably a hairpin loop or a hairpin loopadaptor. Suitable hairpin adaptors can be designed using methods knownin the art. The hairpin loop may be any length. The hairpin loop istypically 110 or fewer nucleotides, such as 100 or fewer nucleotides, 90or fewer nucleotides, 80 or fewer nucleotides, 70 or fewer nucleotides,60 or fewer nucleotides, 50 or fewer nucleotides, 40 or fewernucleotides, 30 or fewer nucleotides, 20 or fewer nucleotides or 10 orfewer nucleotides, in length. The hairpin loop is preferably from about1 to 110, from 2 to 100, from 5 to 80 or from 6 to 50 nucleotides inlength. Longer lengths of the hairpin loop, such as from 50 to 110nucleotides, are preferred if the loop is involved in the differentialselectability of the adaptor. Similarly, shorter lengths of the hairpinloop, such as from 1 to 5 nucleotides, are preferred if the loop is notinvolved in the selectable binding as discussed below.

The hairpin adaptor may be ligated to either end of the first and/orsecond polynucleotide, i.e. the 5′ or the 3′ end. The hairpin adaptormay be ligated to the first and/or second polynucleotide using anymethod known in the art. The hairpin adaptor may be ligated using aligase, such as T4 DNA ligase, E. coli DNA ligase, Taq DNA ligase, TmaDNA ligase and 9^(o)N DNA ligase.

The two strands of the polynucleotide may be separated using any methodknown in the art. For instance, they may be separated by apolynucleotide binding protein or using conditions which favourdehybridisation (examples of conditions which favour dehybridisationinclude, but are not limited to, high temperature, high pH and theaddition of agents that can disrupt hydrogen bonding or base pairing,such as formamide and urea).

The hairpin adaptor preferably comprises a selectable binding moiety.This allows the first and/or second polynucleotide to be purified orisolated. A selectable binding moiety is a moiety that can be selectedon the basis of its binding properties. Hence, a selectable bindingmoiety is preferably a moiety that specifically binds to a surface. Aselectable binding moiety specifically binds to a surface if it binds tothe surface to a much greater degree than any other moiety used in theinvention. In preferred embodiments, the moiety binds to a surface towhich no other moiety used in the invention binds.

Suitable selective binding moieties are known in the art. Preferredselective binding moieties include, but are not limited to, biotin, apolynucleotide sequence, antibodies, antibody fragments, such as Fab andScSv, antigens, polynucleotide binding proteins, poly histidine tailsand GST tags. The most preferred selective binding moieties are biotinand a selectable polynucleotide sequence. Biotin specifically binds to asurface coated with avidins. Selectable polynucleotide sequencesspecifically bind (i.e. hybridise) to a surface coated with homologussequences. Alternatively, selectable polynucleotide sequencesspecifically bind to a surface coated with polynucleotide bindingproteins.

The hairpin adaptor and/or the selectable binding moiety may comprise aregion that can be cut, nicked, cleaved or hydrolysed. Such a region canbe designed to allow the first and/or second polynucleotide to beremoved from the surface to which it is bound following purification orisolation. Suitable regions are known in the art. Suitable regionsinclude, but are not limited to, an RNA region, a region comprisingdesthiobiotin and streptavidin, a disulphide bond and a photocleavableregion.

The double stranded target polynucleotide preferably comprises a leadersequence at the opposite end of the bridging moiety, such as a hairpinloop or hairpin loop adaptor. Leader sequences are discussed in moredetail below.

Round the Corner Sequencing

In a preferred embodiment, a target double stranded polynucleotide isprovided with a bridging moiety, such as a hairpin loop or hairpin loopadaptor, at one end and the method comprises contacting thepolynucleotide with the pore such that both strands of thepolynucleotide move through the pore and taking one or more measurementsas the both strands of the polynucleotide move with respect to the porewherein the measurements are indicative of one or more characteristicsof the strands of the polynucleotide and thereby characterising thetarget double stranded polynucleotide. In another preferred embodiment,a target double stranded polynucleotide is provided with a bridgingmoiety, such as a hairpin loop or hairpin loop adaptor, at one end andthe method comprises contacting the polynucleotide with the pore andexonuclease such that both strands of the polynucleotide are digested toform individual nucleotides. Any of the embodiments discussed aboveequally apply to this embodiment.

Leader Sequence

Before the contacting step in the strand characterisation/sequencingmethod, the method preferably comprises attaching to the polynucleotidea leader sequence which preferentially threads into the pore. The leadersequence facilitates the method of the invention. The leader sequence isdesigned to preferentially thread into the pore and thereby facilitatethe movement of polynucleotide through the pore. The leader sequence canalso be used to link the polynucleotide to the one or more anchors asdiscussed above.

The leader sequence typically comprises a polymer. The polymer ispreferably negatively charged. The polymer is preferably apolynucleotide, such as DNA or RNA, a modified polynucleotide (such asabasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide. Theleader preferably comprises a polynucleotide and more preferablycomprises a single stranded polynucleotide. The leader sequence cancomprise any of the polynucleotides discussed above. The single strandedleader sequence most preferably comprises a single strand of DNA, suchas a poly dT section. The leader sequence preferably comprises the oneor more spacers.

The leader sequence can be any length, but is typically 10 to 150nucleotides in length, such as from 20 to 150 nucleotides in length. Thelength of the leader typically depends on the transmembrane pore used inthe method.

The leader sequence is preferably part of a Y adaptor as defined below.

Double Coupling

The method of the invention may involve double coupling of a doublestranded polynucleotide. In a preferred embodiment, the method of theinvention comprises:

-   -   (a) providing the double stranded polynucleotide with a Y        adaptor at one end and a bridging moiety adaptor, such as a        hairpin loop adaptor, at the other end, wherein the Y adaptor        comprises one or more first anchors for coupling the        polynucleotide to the membrane, wherein the bridging moiety        adaptor comprises one or more second anchors for coupling the        polynucleotide to the membrane and wherein the strength of        coupling of the bridging moiety adaptor to the membrane is        greater than the strength of coupling of the Y adaptor to the        membrane;    -   (b) contacting the polynucleotide provided in step (a) with a        CsgG pore or mutant thereof, such as a pore the invention, such        that the polynucleotide moves with respect to, such as through,        the pore; and    -   (c) taking one or more measurements as the polynucleotide moves        with respect to the pore, wherein the measurements are        indicative of one or more characteristics of the polynucleotide,        and thereby characterising the target polynucleotide.

This type of method is discussed in detail in the UK Application No.1406147.7.

The double stranded polynucleotide is provided with a Y adaptor at oneend and a bridging moiety adaptor at the other end. The Y adaptor and/orthe bridging moiety adaptor are typically polynucleotide adaptors. Theymay be formed from any of the polynucleotides discussed above.

The Y adaptor typically comprises (a) a double stranded region and (b) asingle stranded region or a region that is not complementary at theother end. The Y adaptor may be described as having an overhang if itcomprises a single stranded region. The presence of a non-complementaryregion in the Y adaptor gives the adaptor its Y shape since the twostrands typically do not hybridise to each other unlike the doublestranded portion. The Y adaptor comprises the one or more first anchors.Anchors are discussed in more detail above.

The Y adaptor preferably comprises a leader sequence whichpreferentially threads into the pore. This is discussed above.

The bridging moiety adaptor preferably comprises a selectable bindingmoiety as discussed above. The bridging moiety adaptor and/or theselectable binding moiety may comprise a region that can be cut, nicked,cleaved or hydrolysed as discussed above.

If one or more helicases and one or more molecular brakes are used asdiscussed above, the Y adaptor preferably comprises the one or morehelicases and the bridging moiety adaptor preferably comprises the oneor more molecular brakes.

The Y adaptor and/or the bridging moiety adaptor may be ligated to thepolynucleotide using any method known in the art. One or both of theadaptors may be ligated using a ligase, such as T4 DNA ligase, E. coliDNA ligase, Taq DNA ligase, Tma DNA ligase and 9^(o)N DNA ligase.Alternatively, the adaptors may be added to the polynucleotide using themethods of the invention discussed below.

In a preferred embodiment, step a) of the method comprises modifying thedouble stranded polynucleotide so that it comprises the Y adaptor at oneend and the bridging moiety adaptor at the other end. Any manner ofmodification can be used. The method preferably comprises modifying thedouble stranded polynucleotide in accordance with the invention. This isdiscussed in more detail below. The methods of modification andcharacterisation may be combined in any way.

The strength of coupling (or binding) of the bridging moiety adaptor tothe membrane is greater than the strength of coupling (or binding) ofthe Y adaptor to the membrane. This can be measured in any way. Asuitable method for measuring the strength of coupling (or binding) isdisclosed in the Examples of the UK Application No. 1406147.7.

The strength of coupling (or binding) of the bridging moiety adaptor ispreferably at least 1.5 times the strength of coupling (or binding) ofthe Y adaptor, such as at least twice, at least three times, at leastfour times, at least five or at least ten times the strength of coupling(or binding) of the anchor adaptor. The affinity constant (Kd) of thebridging moiety adaptor for the membrane is preferably at least 1.5times the affinity constant of the Y adaptor, such as at least twice, atleast three times, at least four times, at least five or at least tentimes the strength of coupling of the Y adaptor.

There are several ways in which the bridging moiety adaptor couples (orbinds) more strongly to the membrane than the Y adaptor. For instance,the bridging moiety adaptor may comprise more anchors than the Yadaptor. For instance, the bridging moiety adaptor may comprise 2, 3 ormore second anchors whereas the Y adaptor may comprise one first anchor.

The strength of coupling (or binding) of the one or more second anchorsto the membrane may be greater than the strength of coupling (orbinding) of the one or more first anchors to the membrane. The strengthof coupling (or binding) of the one or more second anchors to thebridging moiety adaptor may be greater than the strength of coupling (orbinding) of the one or more first anchors to the Y adaptor. The one ormore first anchors and the one or more second anchors may be attached totheir respective adaptors via hybridisation and the strength ofhybridisation is greater in the one or more second anchors than in theone or more first anchors. Any combination of these embodiments may alsobe used in the invention. Strength of coupling (or binding) may bemeasure using known techniques in the art.

The one or more second anchors preferably comprise one or more groupswhich couple(s) (or bind(s)) to the membrane with a greater strengththan the one or more groups in the one or more first anchors whichcouple(s) (or bind(s)) to the membrane. In preferred embodiments, thebridging moiety adaptor/one or more second anchors couple (or bind) tothe membrane using cholesterol and the Y adaptor/one or more firstanchors couple (or bind) to the membrane using palmitate. Cholesterolbinds to triblock copolymer membranes and lipid membranes more stronglythan palmitate. In an alternative embodiment, the bridging moietyadaptor/one or more second anchors couple (or bind) to the membraneusing a mono-acyl species, such as palmitate, and the Y adaptor/one ormore first anchors couple (or bind) to the membrane using a diacylspecies, such as dipalmitoylphosphatidylcholine.

Adding Hairpin Loops and Leader Sequences

Before provision, a double stranded polynucleotide may be contacted witha MuA transposase and a population of double stranded MuA substrates,wherein a proportion of the substrates in the population are Y adaptorscomprising the leader sequence and wherein a proportion of thesubstrates in the population are hairpin loop adaptors. The transposasefragments the double stranded polynucleotide analyte and ligates MuAsubstrates to one or both ends of the fragments. This produces aplurality of modified double stranded polynucleotides comprising theleader sequence at one end and the hairpin loop at the other. Themodified double stranded polynucleotides may then be investigated usingthe method of the invention.

Each substrate in the population preferably comprises at least oneoverhang of universal nucleotides such that the transposase fragmentsthe template polynucleotide and ligates a substrate to one or both endsof the double stranded fragments and thereby produces a plurality offragment/substrate constructs and wherein the method further comprisesligating the overhangs to the fragments in the constructs and therebyproducing a plurality of modified double stranded polynucleotides.Suitable universal nucleotides are discussed above. The overhang ispreferably five nucleotides in length.

Alternatively, each substrate in population preferably comprises (i) atleast one overhang and (ii) at least one nucleotide in the same strandas the at least one overhang which comprises a nucleoside that is notpresent in the template polynucleotide such that the transposasefragments the template polynucleotide and ligates a substrate to one orboth ends of the double stranded fragments and thereby produces aplurality of fragment/substrate constructs, and wherein the methodfurther comprises (a) removing the overhangs from the constructs byselectively removing the at least one nucleotide and thereby producing aplurality of double stranded constructs comprising single stranded gapsand (b) repairing the single stranded gaps in the constructs and therebyproducing a plurality of modified double stranded polynucleotides. Thepolynucleotide typically comprises the nucleosides deoxyadenosine (dA),deoxyuridine (dU) and/or thymidine (dT), deoxyguanosine (dG) anddeoxycytidine (dC). The nucleoside that is not present in thepolynucleotide is preferably abasic, adenosine (A), uridine (U),5-methyluridine (m⁵U), cytidine (C) or guanosine (G) or comprises urea,5, 6 dihydroxythymine, thymine glycol, 5-hydroxy-5 methylhydanton,uracil glycol, 6-hydroxy-5, 6-dihdrothimine, methyltartronylurea, 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoedenine, fapy-guanine,methy-fapy-guanine, fapy-adenine, aflatoxin B1-fapy-guanine,5-hydroxy-cytosine, 5-hydroxy-uracil, 3-methyladenine, 7-methylguanine,1,N6-ethenoadenine, hypoxanthine, 5-hydroxyuracil,5-hydroxymethyluracil, 5-formyluracil or a cis-syn-cyclobutanepyrimidine dimer. The at least one nucleotide preferably is 10nucleotides or fewer from the overhang. The at least one nucleotide isthe first nucleotide in the overhang. All of the nucleotides in theoverhang preferably comprise a nucleoside that is not present in thetemplate polynucleotide.

These MuA based methods are disclosed in International Application No.PCT/GB2014/052505. They are also discussed in detail in the UKApplication No. 1406147.7.

One or more helicases may be attached to the MuA substrate Y adaptorsbefore they are contacted with the double stranded polynucleotide andMuA transposase. Alternatively, one or more helicases may be attached tothe MuA substrate Y adaptors before they are contacted with the doublestranded polynucleotide and MuA transposase.

One or more molecular brakes may be attached to the MuA substratehairpin loop adaptors before they are contacted with the double strandedpolynucleotide and MuA transposase. Alternatively, one or more molecularbrakes may be attached to the MuA substrate hairpin loop adaptors beforethey are contacted with the double stranded polynucleotide and MuAtransposase.

Uncoupling

The method of the invention may involve characterising multiple targetpolynucleotides and uncoupling of the at least the first targetpolynucleotide.

In a preferred embodiment, the invention involves characterising two ormore target polynucleotides. The method comprises:

-   -   (a) providing a first polynucleotide in a first sample;    -   (b) providing a second polynucleotide in a second sample;    -   (c) coupling the first polynucleotide in the first sample to a        membrane using one or more anchors;    -   (d) contacting the first polynucleotide with CsgG pore or mutant        thereof, such as a pore of the invention, such that the        polynucleotide moves with respect to, such as through, the pore;    -   (e) taking one or more measurements as the first polynucleotide        moves with respect to the pore wherein the measurements are        indicative of one or more characteristics of the first        polynucleotide and thereby characterising the first        polynucleotide;    -   (f) uncoupling the first polynucleotide from the membrane;    -   (g) coupling the second polynucleotide in the second sample to        the membrane using one or more anchors;    -   (h) contacting the second polynucleotide with the CsgG pore or        mutant thereof, such as a pore of the invention, such that the        second polynucleotide moves with respect to, such as through,        the pore; and    -   (i) taking one or more measurements as the second polynucleotide        moves with respect to the pore wherein the measurements are        indicative of one or more characteristics of the second        polynucleotide and thereby characterising the second        polynucleotide.

This type of method is discussed in detail in the UK Application No.1406155.0.

Step (f) (i.e. uncoupling of the first polynucleotide) may be performedbefore step (g) (i.e. before coupling the second polynucleotide to themembrane). Step (g) may be performed before step (f). If the secondpolynucleotide is coupled to the membrane before the firstpolynucleotide is uncoupled, step (f) preferably comprises selectivelyuncoupling the first polynucleotide from the membrane (i.e. uncouplingthe first polynucleotide but not the second polynucleotide from themembrane). A skilled person can design a system in which selectiveuncoupling is achieved. Steps (f) and (g) may be performed at the sametime. This is discussed in more detail below.

In step (f), at least 10% of the first polynucleotide is preferablyuncoupled from the membrane. For instance, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or at least 95% of the first polynucleotide may be uncoupledfrom the membrane. Preferably, all of the first polynucleotide isuncoupled from the membrane. The amount of the first polynucleotideuncoupled from the membrane can be determined using the pore. This isdisclosed in the Examples.

The first polynucleotide and second polynucleotide may be different fromone another. Alternatively, the first and second polynucleotides may bedifferent polynucleotides. In such instances, there may be no need toremove at least part of the first sample before adding the secondpolynucleotide. This is discussed in more detail below. If the methodconcerns investigating three or more polynucleotides, they may all bedifferent from one another or some of them may be different from oneanother.

The first polynucleotide and the second polynucleotide may be twoinstances of the same polynucleotide. The first polynucleotide may beidentical to the second polynucleotide. This allows proof reading. Ifthe method concerns investigating three or more polynucleotides, theymay all be three or more instances of the same polynucleotide or some ofthem may be separate instances of the same polynucleotide.

The first sample and second sample may be different from one another.For instance, the first sample may be derived from a human and thesecond sample may be derived from a virus. If the first and secondsamples are different from one another, they may contain or be suspectedof containing the same first and second polynucleotides. If the methodconcerns investigating three or more samples, they may all be differentfrom one another or some of them may be different from one another.

The first sample and the second sample are preferably two instances ofthe same sample. The first sample is preferably identical to the secondsample. This allows proof reading. If the method concerns investigatingthree or more samples, they may all be three or more instances of thesame sample or some of them may be separate instances of the samesample.

Any number of polynucleotides can be investigated. For instance, themethod of the invention may concern characterising 3, 4, 5, 6, 7, 8, 9,10, 20, 30, 50, 100 or more polynucleotides. If three or morepolynucleotides are investigated using the method of the invention, thesecond polynucleotide is also uncoupled from the membrane and therequisite number of steps are added for the third polynucleotide. Thesame is true for four or more polynucleotides.

The method of the invention involves uncoupling the first polynucleotidefrom the membrane. The method of the invention may involve uncouplingthe second polynucleotide from the membrane if three or morepolynucleotides are being investigated.

The first polynucleotide can be uncoupled from the membrane using anyknown method. The first polynucleotide is preferably not uncoupled fromthe membrane in step (f) using the transmembrane pore. The firstpolynucleotide is preferably not uncoupled from the membrane using avoltage or an applied potential.

Step (f) preferably comprises uncoupling the first polynucleotide fromthe membrane by removing the one or more anchors from the membrane. Ifthe anchors are removed, the second polynucleotide is coupled to themembrane using other (or separate) anchors. The anchors used to couplethe second polynucleotide may be the same type of anchors used to couplethe first polynucleotide or different type of anchors.

Step (f) more preferably comprises contacting the one or more anchorswith an agent which has a higher affinity for the one or more anchorsthan the anchors have for the membrane. A variety of protocols forcompetitive binding or immunoradiometric assays to determine thespecific binding capability of molecules are well known in the art (seefor example Maddox et ael, J. Exp. Med. 158, 1211-1226, 1993). The agentremoves the anchor(s) from the membrane and thereby uncouples the firstpolynucleotide. The agent is preferably a sugar. Any sugar which bindsto the one or more anchors with a higher affinity than the one or moreanchors have for the membrane may be used. The sugar may be acyclodextrin or derivative thereof as discussed below.

If one or more anchors comprise a hydrophobic anchor, such ascholesterol, the agent is preferably a cyclodextrin or a derivativethereof or a lipid. The cyclodextrin or derivative thereof may be any ofthose disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am.Chem. Soc. 116, 6081-6088. The agent is more preferablyheptakis-6-amino-β-cyclodextrin (am₇-βCD),6-monodeoxy-6-monoamino-3-cyclodextrin (am₁-□CD) orheptakis-(6-deoxy-6-guanidino)cyclodextrin (gu₇-βCD). Any of the lipidsdisclosed herein may be used.

If an anchor comprise(s) streptavidin, biotin or desthiobiotin, theagent is preferably biotin, desthiobiotin or streptavidin. Both biotinand desthiobiotin bind to streptavidin with a higher affinity thanstreptavidin binds to the membrane and vice versa. Biotin has a strongeraffinity for streptavidin than desthiobiotin. An anchor comprisingstreptavidin may therefore be removed from the membrane using biotin orstreptavidin and vice versa.

If an anchor comprises a protein, the agent is preferably an antibody orfragment thereof which specifically binds to the protein. An antibodyspecifically binds to a protein if it binds to the protein withpreferential or high affinity, but does not bind or binds with only lowaffinity to other or different proteins. An antibody binds withpreferential or high affinity if it binds with a Kd of 1×10⁻⁶ M or less,more preferably 1×10⁻⁷ M or less, 5×10⁻⁸ M or less, more preferably1×10⁻⁸ M or less or more preferably 5×10⁻⁹ M or less. An antibody bindswith low affinity if it binds with a Kd of 1×10⁻⁶ M or more, morepreferably 1×10⁻⁵ M or more, more preferably 1×10⁻⁴ M or more, morepreferably 1×10⁻³ M or more, even more preferably 1×10⁻² M or more. Anymethod may be used to detect binding or specific binding. Methods ofquantitatively measuring the binding of an antibody to a protein arewell known in the art. The antibody may be a monoclonal antibody or apolyclonal antibody. Suitable fragments of antibodies include, but arenot limited to, Fv, F(ab′) and F(ab′)₂ fragments, as wel as single chainantibodies. Furthermore, the antibody or fragment thereof may be achimeric antibody or fragment thereof, a CDR-grafted antibody orfragment thereof or a humanised antibody or fragment thereof.

Step (f) preferably comprises contacting the one or more anchors with anagent which reduces ability of the one or more anchors to couple to themembrane. For instance, the agent could interfere with the structureand/or hydrophobicity of the one or more anchors and thereby reducetheir ability to couple to the membrane. If an anchor comprisescholesterol, the agent is preferably cholesterol dehydrogenase. If ananchor comprises a lipid, the agent is preferably a phospholipase. If ananchor comprises a protein, the agent is preferably a proteinase orurea. Other combination of suitable anchors and agents will be clear toa person skilled in the art.

Step (f) preferably comprises uncoupling the first polynucleotide fromthe membrane by separating the first polynucleotide from the one or moreanchors. This can be done in any manner. For instance, the linker couldbe cut in an anchor comprising a linker. This embodiment is particularlyapplicable to anchors which involve linkage via hybridisation. Suchanchors are discussed above.

Step (f) more preferably comprises uncoupling the first polynucleotidefrom the membrane by contacting the first polynucleotide and the one ormore anchors with an agent which competes with the first polynucleotidefor binding to one or more anchors. Methods for determining andmeasuring competitive binding are known in the art. The agent ispreferably a polynucleotide which competes with the first polynucleotidefor hybridisation to the one or more anchors. For instance, if the firstpolynucleotide is coupled to the membrane using one or more anchorswhich involve hybridisation, the polynucleotide can be uncoupled bycontacting the one or more anchors with a polynucleotide which alsohybridises to the site of hybridisation. The polynucleotide agent istypically added at a concentration that is higher than the concentrationof the first polynucleotide and one or more anchors. Alternatively, thepolynucleotide agent may hybridise more strongly to the one or moreanchors than the first polynucleotide.

Step (f) more preferably comprises (i) contacting the firstpolynucleotide and the one or more anchors with urea,tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), streptavidinor biotin, UV light, an enzyme or a binding agent; (ii) heating thefirst polynucleotide and the one or more anchors; or (iii) altering thepH. Urea, tris(2-carboxyethyl)phosphine (TCEP) or dithiothreitol (DTT)are capable of disrupting anchors and separating the firstpolynucleotide from the membrane. If an anchor comprises astreptavidin-biotin link, then a streptavidin agent will compete forbinding to the biotin. If an anchor comprises astreptavidin-desthiobiotin link, then a biotin agent will compete forbinding to the streptavidin. UV light can be used to breakdownphotolabile groups. Enzymes and binding agents can be used to cut,breakdown or unravel the anchor. Preferred enzymes include, but are notlimited to, an exonuclease, an endonuclease or a helicase. Preferredbinding agents include, but are not limited to, an enzyme, an antibodyor a fragment thereof or a single-stranded binding protein (SSB). Any ofthe enzymes discussed below or antibodies discussed above may be used.Heat and pH can be used to disrupt hybridisation and other linkages.

If the first polynucleotide is uncoupled from the membrane by separatingthe first polynucleotide from the one or more anchors, the one or moreanchors will remain in the membrane. Step (g) preferably comprisescoupling the second polynucleotide to the membrane using the one or moreanchors that was separated from the first polynucleotide. For instance,the second polynucleotide may also be provided with one or morepolynucleotides which hybridise(s) to the one or more anchors thatremain in the membrane. Alternatively, step (g) preferably comprisescoupling the second polynucleotide to the membrane using one or moreseparate anchors from the ones separated from the first polynucleotide(i.e. one or more other anchors). The one or more separate anchors maybe the same type of anchors used to couple the first polynucleotide tothe membrane or may be different types of anchors. Step (g) preferablycomprises coupling the second polynucleotide to the membrane using oneor more different anchors from the one or more anchors separated fromthe first polynucleotide.

In a preferred embodiment, steps (f) and (g) comprise uncoupling thefirst polynucleotide from the membrane by contacting the membrane withthe second polynucleotide such that the second polynucleotide competeswith the first polynucleotide for binding to the one or more anchors andreplaces the first polynucleotide. For instance, if the firstpolynucleotide is coupled to the membrane using one or more anchorswhich involve hybridisation, the first polynucleotide can be uncoupledby contacting the anchors with the second polynucleotide attached topolynucleotides which also hybridise to the sites of hybridisation inthe one or more anchors. The second polynucleotide is typically added ata concentration that is higher than the concentration of the firstpolynucleotide and the one or more anchors. Alternatively, the secondpolynucleotide may hybridise more strongly to the one or more anchorsthan the first polynucleotide.

Removal or Washing

Although the first polynucleotide is uncoupled from the membrane in step(f), it is not necessarily removed or washed away. If the secondpolynucleotide can be easily distinguished from the firstpolynucleotide, there is no need to remove the first polynucleotide.

Between steps (f) and (g), the method preferably further comprisesremoving at least some of the first sample from the membrane. At least10% of the first sample may be removed, such as at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80% or at least 90% of the first sample may be removed.

The method more preferably further comprises removing all of the firstsample from the membrane. This can be done in any way. For instance, themembrane can be washed with a buffer after the first polynucleotide hasbeen uncoupled. Suitable buffers are discussed below.

Modified Polynucleotides

Before characterisation, a target polynucleotide may be modified bycontacting the polynucleotide with a polymerase and a population of freenucleotides under conditions in which the polymerase forms a modifiedpolynucleotide using the target polynucleotide as a template, whereinthe polymerase replaces one or more of the nucleotide species in thetarget polynucleotide with a different nucleotide species when formingthe modified polynucleotide. The modified polynucleotide may then beprovided with one or more helicases attached to the polynucleotide andone or more molecular brakes attached to the polynucleotide. This typeof modification is described in UK Application No. 1403096.9. Any of thepolymerases discussed above may be used. The polymerase is preferablyKlenow or 9o North.

The template polynucleotide is contacted with the polymerase underconditions in which the polymerase forms a modified polynucleotide usingthe template polynucleotide as a template. Such conditions are known inthe art. For instance, the polynucleotide is typically contacted withthe polymerase in commercially available polymerase buffer, such asbuffer from New England Biolabs®. The temperature is preferably from 20to 37° C. for Klenow or from 60 to 75° C. for 9o North. A primer or a 3′hairpin is typically used as the nucleation point for polymeraseextension.

Characterisation, such as sequencing, of a polynucleotide using atransmembrane pore typically involves analyzing polymer units made up ofk nucleotides where k is a positive integer (i.e. ‘k-mers’). This isdiscussed in International Application No. PCT/GB2012/052343 (publishedas WO 20131041878). While it is desirable to have clear separationbetween current measurements for different k-mers, it is common for someof these measurements to overlap. Especially with high numbers ofpolymer units in the k-mer, i.e. high values of k, it can becomedifficult to resolve the measurements produced by different k-mers, tothe detriment of deriving information about the polynucleotide, forexample an estimate of the underlying sequence of the polynucleotide.

By replacing one or more nucleotide species in the target polynucleotidewith different nucleotide species in the modified polynucleotide, themodified polynucleotide contains k-mers which differ from those in thetarget polynucleotide. The different k-mers in the modifiedpolynucleotide are capable of producing different current measurementsfrom the k-mers in the target polynucleotide and so the modifiedpolynucleotide provides different information from the targetpolynucleotide. The additional information from the modifiedpolynucleotide can make it easier to characterise the targetpolynucleotide. In some instances, the modified polynucleotide itselfmay be easier to characterise. For instance, the modified polynucleotidemay be designed to include k-mers with an increased separation or aclear separation between their current measurements or k-mers which havea decreased noise.

The polymerase preferably replaces two or more of the nucleotide speciesin the target polynucleotide with different nucleotide species whenforming the modified polynucleotide. The polymerase may replace each ofthe two or more nucleotide species in the target polynucleotide with adistinct nucleotide species. The polymerase may replace each of the twoor more nucleotide species in the target polynucleotide with the samenucleotide species.

If the target polynucleotide is DNA, the different nucleotide species inthe modified typically comprises a nucleobase which differs fromadenine, guanine, thymine, cytosine or methylcytosine and/or comprises anucleoside which differs from deoxyadenosine, deoxyguanosine, thymidine,deoxycytidine or deoxymethylcytidine. If the target polynucleotide isRNA, the different nucleotide species in the modified polynucleotidetypically comprises a nucleobase which differs from adenine, guanine,uracil, cytosine or methylcytosine and/or comprises a nucleoside whichdiffers from adenosine, guanosine, uridine, cytidine or methylcytidine.The different nucleotide species may be any of the universal nucleotidesdiscussed above.

The polymerase may replace the one or more nucleotide species with adifferent nucleotide species which comprises a chemical group or atomabsent from the one or more nucleotide species. The chemical group maybe a propynyl group, a thio group, an oxo group, a methyl group, ahydroxymethyl group, a formyl group, a carboxy group, a carbonyl group,a benzyl group, a propargyl group or a propargylamine group.

The polymerase may replace the one or more nucleotide species with adifferent nucleotide species which lacks a chemical group or atompresent in the one or more nucleotide species. The polymerase mayreplace the one or more of the nucleotide species with a differentnucleotide species having an altered electronegativity. The differentnucleotide species having an altered electronegativity preferablycomprises a halogen atom.

The method preferably further comprises selectively removing thenucleobases from the one or more different nucleotides species in themodified polynucleotide.

Analyte Delivery

The target analyte is preferably attached to a microparticle whichdelivers the analyte towards the membrane. This type of delivery isdisclosed in UK Application No. 1418469.1.

Any type of microparticle and attachment method may be used.

Other Characterisation Method

In another embodiment, a polynucleotide is characterised by detectinglabelled species that are added to the target polynucleotide by apolymerase and then released. The polymerase uses the polynucleotide asa template. Each labelled species is specific for each nucleotide. Thepolynucleotide is contacted with a CsgG pore or mutant thereof, such asa pore of the invention, a polymerase and labeled nucleotides such thatphosphate labelled species are sequentially added to the thepolynucleotide by the polymerase, wherein the phosphate species containa label specific for each nucleotide. The labelled species may bedetected using the pore before they are released from the nucleotides(i.e. as they are added to the target polynucleotide) or after they arereleased from the nucleotides.

The polymerase may be any of those discussed above. The phosphatelabelled species are detected using the pore and thereby characterisingthe polynucleotide. This type of method is disclosed in EuropeanApplication No. 13187149.3 (published as EP 2682460). Any of theembodiments discussed above equally apply to this method.

Examples of labelled species include, but are not limited to, polymers,polyethylene glycols, sugars, cyclodextrins, fluorophores, drugs,metabolites, peptides. A non-limiting example of such tags can be foundin the work of Kumar et al. Sci Rep. 2012; 2:684. Epub 2012 Sep. 21.

Methods of Forming Sensors

The invention also provides a method of forming a sensor forcharacterising a target polynucleotide. The method comprises forming acomplex between a CsgG pore or mutant thereof, such as a pore of theinvention, and a polynucleotide binding protein, such as a helicase oran exonuclease. The complex may be formed by contacting the pore and theprotein in the presence of the target polynucleotide and then applying apotential across the pore. The applied potential may be a chemicalpotential or a voltage potential as described above. Alternatively, thecomplex may be formed by covalently attaching the pore to the protein.Methods for covalent attachment are known in the art and disclosed, forexample, in International Application Nos. PCT/GB09/001679 (published asWO 2010/004265) and PCT/GB10/000133 (published as WO 2010/086603). Thecomplex is a sensor for characterising the target polynucleotide. Themethod preferably comprises forming a complex between a CsgG pore ormutant thereof, such as a pore of the invention, and a helicase. Any ofthe embodiments discussed above equally apply to this method.

The invention also provides a sensor for characterising a targetpolynucleotide. The sensor comprises a complex between a CsgG pore ormutant thereof, such as a pore of the invention, and a polynucleotidebinding protein. Any of the embodiments discussed above equally apply tothe sensor of the invention.

Kits

The present invention also provides a kit for characterising a targetpolynucleotide. The kit comprises a CsgG pore or mutant thereof, such asa pore of the invention, and the components of a membrane. The membraneis preferably formed from the components. The pore is preferably presentin the membrane. The kit may comprise components of any of the membranesdisclosed above, such as an amphiphilic layer or a triblock copolymermembrane.

The kit may further comprise a polynucleotide binding protein. Any ofthe polynucleotide binding proteins discussed above may be used.

The kit may further comprise one or more anchors for coupling thepolynucleotide to the membrane.

The kit is preferably for characterising a double strandedpolynucleotide and preferably comprises a Y adaptor and a hairpin loopadaptor. The Y adaptor preferably has one or more helicases attached andthe hairpin loop adaptor preferably has one or more molecular brakesattached. The Y adaptor preferably comprises one or more first anchorsfor coupling the polynucleotide to the membrane, the hairpin loopadaptor preferably comprises one or more second anchors for coupling thepolynucleotide to the membrane and the strength of coupling of thehairpin loop adaptor to the membrane is preferably greater than thestrength of coupling of the Y adaptor to the membrane.

The kit of the invention may additionally comprise one or more otherreagents or instruments which enable any of the embodiments mentionedabove to be carried out. Such reagents or instruments include one ormore of the following: suitable buffer(s) (aqueous solutions), means toobtain a sample from a subject (such as a vessel or an instrumentcomprising a needle), means to amplify and/or express polynucleotides orvoltage or patch clamp apparatus. Reagents may be present in the kit ina dry state such that a fluid sample resuspends the reagents. The kitmay also, optionally, comprise instructions to enable the kit to be usedin the method of the invention or details regarding for which organismthe method may be used.

Apparatus

The invention also provides an apparatus for characterising a targetanalyte, such as a target polynucleotide. The apparatus comprises aplurality of CsgG pores or mutants thereof and a plurality of membranes.The plurality of pores are preferably present in the plurality ofmembranes. The number of pores and membranes is preferably equal.Preferably, a single pore is present in each membrane.

The apparatus preferably further comprises instructions for carrying outthe method of the invention. The apparatus may be any conventionalapparatus for analyte analysis, such as an array or a chip. Any of theembodiments discussed above with reference to the methods of theinvention are equally applicable to the apparatus of the invention. Theapparatus may further comprise any of the features present in the kit ofthe invention.

The apparatus is preferably set up to carry out the method of theinvention.

The apparatus preferably comprises:

-   -   a sensor device that is capable of supporting the plurality of        pores and membranes and being operable to perform analyte        characterisation using the pores and membranes; and    -   at least one port for delivery of the material for performing        the characterisation.

Alternatively, the apparatus preferably comprises:

-   -   a sensor device that is capable of supporting the plurality of        pores and membranes being operable to perform analyte        characterisation using the pores and membranes; and    -   at least one reservoir for holding material for performing the        characterisation.

The apparatus more preferably comprises:

-   -   a sensor device that is capable of supporting the membrane and        plurality of pores and membranes and being operable to perform        analyte characterising using the pores and membranes;    -   at least one reservoir for holding material for performing the        characterising;    -   a fluidics system configured to controllably supply material        from the at least one reservoir to the sensor device; and    -   one or more containers for receiving respective samples, the        fluidics system being configured to supply the samples        selectively from one or more containers to the sensor device.

The apparatus may be any of those described in International ApplicationNo. No. PCT/GB08/004127 (published as WO 2009/077734), PCT/GB10/000789(published as WO 2010/122293), International Application No.PCT/GB10/002206 (published as WO 2011/067559) or InternationalApplication No. PCT/US99/25679 (published as WO 00/28312).

The following Examples Illustrate the invention.

EXAMPLES Example 1: Cloning and Strains of CsaG

Expression constructs for the production of outer membrane localizedC-terminally StrepII-tagged CsgG (pPG1) and periplasmic C-terminallyStrepII-tagged CsgG_(C1S) (pPG2) have been described (Goyal, P. et al.,Acta Crystallogr. F. Struct. Biol. Cryst. Commun. 2013, 69, 1349-1353).For selenomethionine labeling, StrepII-tagged CsgG_(C1S) was expressedin the cytoplasm because of increased yields. Therefore, pPG2 wasaltered to remove the N-terminal signal peptide using inverse PCR withprimers 5′-TCT TTA AC CGC CCC GCC TAA AG-3′ (forward) (SEQ ID NO: 437)and 5-CAT TTT TTG CCC TCG TTA TC-3′ (reverse) (pPG3) (SEQ ID NO: 438).For phenotypic assays, a csgG deletion mutant of E. coli BW25141 (E.coli NVG2) was constructed by the method described [Datsenko, K. A. etal, Proc. Natl Acad. Sci. USA 97, 6640-6645 (2000)] (with primers 5′-AATAAC TCA ACC GAT TTT TAA GCC CCA GCT TCA TAA GGA AAA TAA TCG TGT AGG CTGGAG CTG CTT C-3′ (SEQ ID NO: 439) and 5′-CGC TTA AAC AGT AAA ATG CCG GATGAT AAT TCC GGC TTT TTT ATC TGC ATA TGA ATA TCC TCC TTA G-3′ (SEQ ID NO:440)). The various CsgG substitution mutants used for Cys accessibilityassays and for phenotypic probing of the channel constriction wereconstructed by site-directed mutagenesis (QuikChange protocol;Stratagene) starting from pMC2, a pTRC99a vector containing csgG undercontrol of the trc promoter (Robinson, L. S., et al, Mol. Microbiol.,2006, 59, 870-881).

Example 2: Protein Expression and Purification

CsgG and CsgG_(C1S) were expressed and purified as described (Robinson,L. S., et al, Mol. Microbiol., 2006, 59, 870-881). In brief, CsgG wasrecombinantly produced in E. coli BL 21 (DE3) transformed with pPG1 andextracted from isolated outer membranes with the use of 1%n-dodecyl-β-D-maltoside (DDM) in buffer A (50 mM Tris-HCl pH 8.0, 500 mMNaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT)). StrepII-tagged CsgG wasloaded onto a 5 ml Strep-Tactin Sepharose column (Iba GmbH) anddetergent-exchanged by washing with 20 column volumes of buffer Asupplemented with 0.5% tetraethylene glycol monooctyl ether (C8E4;Affymetrix) and 4 mM lauryldimethylamine-N-oxide (LDAO; Affymetrix). Theprotein was eluted by the addition of 2.5 mM D-desthiobiotin andconcentrated to 5 mg ml⁻¹ for crystallization experiments. Forselenomethionine labelling, CsgG_(C1S) was produced in the Metauxotrophic strain B834 (DE3) transformed with pPG3 and grown on M9minimal medium supplemented with 40 mg l⁻¹ L-selenomethionine. Cellpellets were resuspended in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mMEDTA, 5 mM DTT, supplemented with cOmplete Protease Inhibitor Cocktail(Roche) and disrupted by passage through a TS series cell disrupter(Constant Systems Ltd) operated at 20×10³ lb in⁻². Labelled CsgG_(C1S)was purified as described (Robinson, L. S., et al, Mol. Microbiol.,2006, 59, 870-881). DTT (5 mM) was added throughout the purificationprocedure to avoid oxidation of selenomethionine.CsgE was produced in E. coli NEBC2566 cells harbouring pNH27 (Nenninger,A. A. et al., Mol. Microbiol. 2011, 81, 486-499). Cell lysates in 25 mMTris-HCl pH 8.0, 150 mM NaCl, 25 mM imidazole, 5% (v/v) glycerol wereloaded on a HisTrap FF (GE Healthcare). CsgE-his was eluted with alinear gradient to 500 mM imidazole in 20 mM Tris-HCl pH 8.0, 150 mMNaCl, 5% (v/v) glycerol buffer. Fractions containing CsgE weresupplemented with 250 mM (NH₄)₂SO₄ and applied to a 5 ml HiTrap PhenylHP column (GE Healthcare) equilibrated with 20 mM Tris-HCl pH 8.0, 100mM NaCl, 250 mM (NH₄)₂SO₄, 5% (v/v) glycerol. A linear gradient to 20 mMTris-HCl pH 8.0, 10 mM NaCl, 5% (v/v) glycerol was applied for elution.CsgE containing fractions were loaded onto a Superose 6 Prep Grade10/600 (GE Healthcare) column equilibrated in 20 mM Tris-HCl pH 8.0, 100mM NaCl, 5% (v/v) glycerol.

Example 3: In-Solution Oligomeric State Assessment

About 0.5 mg each of detergent-solubilized CsgG (0.5% C8E4, 4 mM LDAO)and CsgG_(C1S) were applied to a Superdex 200 10/300 GL analytical gelfiltration column (GE Healthcare) equilibrated with 25 mM Tris-HCl pH8.0, 500 mM NaCl, 1 mM DTT, 4 mM LDAO and 0.5% C8E4 (CsgG) or with 25 mMTris-HCl pH 8.0, 200 mM NaCl (CsgG_(C1S)), and run at 0.7 ml min⁻¹. Thecolumn elution volumes were calibrated with bovine thyroglobulin, bovineγ-globulin, chicken ovalbumin, horse myoglobulin and vitamin B₁₂(Bio-Rad) (FIG. 7). Membrane-extracted CsgG, 20 μg of thedetergent-solubilized protein was also run on 3-10% blue native PAGEusing the procedure described in Swamy, M., et al., Sci. STKE 2006, p14, [http://dx.doi.org/10.1126/stke.3452006pl4(2006)] (FIG. 7).NativeMark (Life Technologies) unstained protein standard (7 μl) wasused for molecular mass estimation. Mature CsgG is predominantly foundas discrete nonameric poreforming particles with C9 symmetry, as well astail-to-tail dimers of nonameric pores (i.e. octadecamers with D9symmetry). For the purpose of nanopore sensing applications, a preferredstate of the proteins is a single nonameric pore. The population ofnonameric versus D9 octadecameric pores can be increased by heathingsamples prior to size exclusion chromatography and/or insertion in alipid bilayer for nanopore sensing applications.

Example 4: Crystallization, Data Collection and Structure Determination

Selenomethionine-labelled CsgG_(C1S) was concentrated to 3.8 mg m⁻¹ andcrystallized by sitting-drop vapour diffusion against a solutioncontaining 100 mM sodium acetate pH 4.2, 8% PEG 4000 and 100 mM sodiummalonate pH 7.0. Crystals were incubated in crystallization buffersupplemented with 15% glycerol and flash-frozen in liquid nitrogen.Detergent-solubilized CsgG was concentrated to 5 mg ml⁻¹ andcrystallized by hanging-drop vapour diffusion against a solutioncontaining 100 mM Tris-HCl pH 8.0, 8% PEG 4000, 100 mM NaCl and 500 mMMgCl₂. Crystals were flash-frozen in liquid nitrogen and cryoprotectedby the detergent present in the crystallization solution. Foroptimization of crystal conditions and screening for crystals with gooddiffraction quality, crystals were analysed on beamlines Proxima-1 andProxima-2a (Soleil, France), PX-I (Swiss Light Source, Switzerland),I02, I03, I04 and I24 (Diamond Light Source, UK) and ID14eh2, ID23eh1and ID23eh2 (ESRF, France). Final diffraction data used for structuredetermination of CsgG_(C1S) and CsgG were collected at beamlines I04 andI03, respectively (Table 5).

TABLE 5 Data collection and refinement statistics CsgG_(C1S) CsgG Datacollection Space group P1 C2 Cell dimensions a, b, c (Å) 101.3, 103.6,141.7 161.9, 372.8, 161.9 α, β, γ (°) 111.3, 90.5, 118.2 90.0, 92.9,90.0 Resolution (Å)*  30-2.8 (2.9.2.8) 30-3.6 (3.7-3.6)   30-3.6 (a*),−3.7 (b*), −3.8 (c*)† Rmeas* 15.1 (81.8) 16.2 (90.6)† I/σI* 9.82 (2.03)6.80 (1.89)† Completeness 98.7 (98.3) 91.57 (27.26)  (%)* 99.9 (99.1)†Redundancy* 11.2 (7.0)  4.4 (4.3)  Wilson B (Å²) 46.7 101.0 RefinementResolution (Å)*  30-2.8 (2.9-2.8) 30-3.6 (3.7-3.6)   30-3.6 (a*), −3.7(b*), −3.8 (c*)† No. reflections* 112419 (11159)  102130 (11094) R_(work)R_(free) 0.1881/0.2337 0.3024/0.3542 No. atoms Protein 2885334165 Ligand/ion 0 0 Water 0 0 B-factors (Å²) Protein 57.3 116.7Ligand/ion Water R.m.s deviations Bond lengths (Å) 0.01 0.03 Bond angles(°) 1.31 1.87 Data statistics for CsgG_(C1S) and membrane-extractedCsgG, collected from a single crystal each. *Highest resolution shell isshown in parenthesis. †Values corrected for anisotropic truncation alongreciprocal directions a*, b* and c*.Diffraction data for CsgG_(C1S) were processed using Xia2 and the XDSpackage (Winter, G., J. Appl. Cryst., 2010, 43, 186-190; Kabsch, W.,Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 125-132). Crystals ofCsgG_(C1S) belonged to space group P1 with unit cell dimensions ofa=101.3 Å, b=103.6 Å, c=141.7 Å, α=111.3°, β=90.5°, γ=118.2°, containing16 protein copies in the asymmetric unit. For structure determinationand refinement, data collected at 0.9795 Å wavelength were truncated at2.8 Å on the basis of an //σ/ cutoff of 2 in the highest-resolutionshell. The structure was solved using experimental phases calculatedfrom a single anomalous dispersion (SAD) experiment. A total of 92selenium sites were located in the asymmetric unit by using ShelxC andShelxD (Sheidrick, G. M., Acta Crystallogr. D Biol. Crystallogr. 2010,66, 479-485), and were refined and used for phase calculation with Sharp(Bricogne, G., Acta Crystallogr. D Biol. Crystallogr. 2003, 59,2023-2030) (phasing power 0.79, Figure of merit (FOM) 0.25).Experimental phases were density modified and averaged bynon-crystallographic symmetry (NCS) using Parrot (Cowtan, K., ActaCrystallogr. D Biol. Crystallogr. 2010, 66, 470-478 (FIG. 7; FOM 0.85).An initial model was built with Buccaneer (Cowtan, K., Acta Crystallogr.D Biol. Crystallogr. 2006, 62, 1002-1011) and refined by iterativerounds of maximum-likelihood refinement with Phenix refine (Adams, P. D.et al. Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 213-221) andmanual inspection and model (re)building in Coot (Emsley, P. et al.,Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 486-501). The finalstructure contained 28,853 atoms in 3,700 residues belonging to 16CsgG_(C1S) chains (FIG. 7), with a molprobity (Davis, I. W. et al.,Nucleic Acids Res. 35 (Suppl 2), W375-W383) score of 1.34; 98% of theresidues lay in favoured regions of the Ramachandran plot (99.7% inallowed regions). Electron density maps showed no unambiguous densitycorresponding to possible solvent molecules, and no water molecules orions were therefore built in. Sixteenfold NCS averaging was maintainedthroughout refinement, using strict and local NCS restraints in earlyand late stages of refinement, respectively.Diffraction data for CsgG were collected from a single crystal at 0.9763Å wavelength and were indexed and scaled, using the XDS package (Winter,G., J. Appl. Cryst. 2010, 43, 186-190; Kabsch, W., Acta Crystallogr. DBiol. Crystallogr. 2010, 66, 125-132), in space group C2 with unit-celldimensions a=161.7 Å, b=372.3 Å, c=161.8 Å and β=92.9°, encompassing 18CsgG copies in the asymmetric unit and a 72% solvent content.Diffraction data for structure determination and refinement wereelliptically truncated to resolution limits of 3.6 Å, 3.7 Å and 3.8 Åalong reciprocal cell directions a*, b* and c* and scaledanisotropically with the Diffraction Anisotropy Server (Strong, M. etal., Proc. Natl Acad. Sci. USA 2006, 103, 8060-8065). Molecularreplacement using the CsgG_(C1S) monomer proved unsuccessful. Analysisof the self rotation function revealed D₉ symmetry in the asymmetricunit (not shown). On the basis of on the CsgG_(C1S) structure, anonameric search model was generated in the assumption that after goingfrom a C₈ to C₉ oligomer, the interprotomer arc at the particlecircumference would stay approximately the same as the interprotomerangle changed from 45° to 40°, giving a calculated increase in radius ofabout 4 Å. Using the calculated nonamer as search model, a molecularreplacement solution containing two copies was found with Phaser (McCoy,A. J. et al., J. Appl. Cryst. 2007, 40, 658-674). Inspection ofdensity-modified and NCS-averaged electron density maps (Parrot [Cowtan,K., Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 470-478]; FIG. 8)allowed manual building of the TM1 and TM2 and remodelling of adjacentresidues in the protein core, as well as the building of residues 2-18,which were missing from the CsgG_(C1S) model and linked the α1 helix tothe N-terminal lipid anchor. Refinement of the CsgG model was performedwith Buster-TNT (Smart, O. S. et al., Acta Crystallogr. D Biol.Crystallogr. 2012, 68, 368-380) and Refmac5 (Murshudov, G. N. et al.,Acta Crystallogr. D Biol. Crystallogr., 2011, 67, 355-367) for initialand final refinement rounds, respectively. Eighteenfold local NCSrestraints were applied throughout refinement, and Refmac5 was runemploying a jelly-body refinement with sigma 0.01 and hydrogen-bondrestraints generated by Prosmart (Nicholls, R. A., Long, F. & Murshudov,G. N., Acta Crystallogr. D Biol. Crystallogr. 2011, 68, 404-417). Thefinal structure contained 34,165 atoms in 4,451 residues belonging to 18CsgG chains (FIG. 7), with a molprobity score of 2.79; 93.0% of theresidues lay in favoured regions of the Ramachandran plot (99.3% inallowed regions). No unambiguous electron density corresponding theN-terminal lipid anchor could be discerned.

Example 5: Congo Red Assay

For analysis of Congo red binding, a bacterial overnight culture grownat 37° C. in Lysogeny Broth (LB) was diluted in LB medium until a D₆₀₀of 0.5 was reached. A 5 μl sample was spotted on LB agar platessupplemented with ampicillin (100 mg l⁻¹), Congo red (100 mg l⁻¹) and0.1% (w/v) isopropyl β-D-thiogalactoside (IPTG). Plates were incubatedat room temperature (20 to 22° C.) for 48 h to induce curli expression.The development of the colony morphology and dye binding were observedat 48 h.

Example 6: Cysteine Accessibility Assays

Cysteine mutants were generated in pMC2 using site-directed mutagenesisand expressed in E. coli LSR12 (Chapman, M. R. et al., Science, 2002,295, 851-855). Bacterial cultures grown overnight were spotted onto LBagar plates containing 1 mM IPTG and 100 mg l⁻¹ ampicillin. Plates wereincubated at room temperature and cells were scraped after 48 h,resuspended in 1 ml of PBS and normalized using D₆₀₀. The cells werelysed by sonication and centrifuged for 20 s at 3,000 g at 4° C. toremove unbroken cells from cell lysate and suspended membranes. Proteinsin the supernatant were labelled with 15 mM methoxypolyethyleneglycol-maleimide (MAL-PEG 5 kDa) for 1 h at room temperature. Thereaction was stopped with 100 mM DTT and centrifuged at 40,000 r.p.m.(˜100,000 g) in a 50.4 Ti rotor for 20 min at 4° C. to pellet totalmembranes. The pellet was washed with 1% sodium lauroyl sarcosinate tosolubilize cytoplasmic membranes and centrifuged again. The resultingouter membranes were resuspended and solubilized using PBS containing 1%DDM. Metal-affinity pulldowns with nickel beads were used for SDS-PAGEand anti-His western blots. E. coli LSR12 cells with empty pMC2 vectorwere used as negative control.

Example 7: ATR-FTIR Spectroscopy

ATR-FTIR measurements were performed on an Equinox 55 infraredspectrophotometer (Bruker), continuously purged with dried air, equippedwith a liquid-nitrogen-refrigerated mercury cadmium telluride detectorand a Golden Gate reflectance accessory (Specac). The internalreflection element was a diamond crystal (2 mm×2 mm) and the beamincidence angle was 45°. Each purified protein sample (1 μl) was spreadat the surface of the crystal and dried under a gaseous nitrogen flow toform a film. Each spectrum, recorded at 2 cm⁻¹ resolution, was anaverage of 128 accumulations for improved signal-to-noise ratio. All thespectra were treated with water vapour contribution subtraction,smoothed at a final resolution of 4 cm⁻¹ by apodization and normalizedon the area of the Amide I band (1,700-1,600 cm⁻¹) to allow theircomparison (Goormaghtigh, E.; Ruysschaert, J. M., Spectrochim. Acta,1994, 50A, 2137-2144).

Example 8: Negative Stain EM and Symmetry Determination

Negative stain EM was used to monitor in-solution oligomerization statesof CsgG, CsgG_(C1S) and CsgE. CsgE, CsgG_(C1S) and amphipol-bound CsgGwere adjusted to a concentration of 0.05 mg ml⁻¹ and applied toglow-discharged carbon-coated copper grids (CF-400; Electron MicroscopySciences). After 1 min incubation, samples were blotted, then washed andstained in 2% uranyl acetate. Images were collected on a Tecnai T12BioTWIN LaB6 microscope operating at a voltage of 120 kV, at amagnification of ×49,000 and defocus between 800 and 2,000 nm. Contrasttransfer function (CTF), phase flipping and particle selection wereperformed as described for cryo-EM. For membrane-extracted CsgG,octadecameric particles (1,780 in all) were analysed separately fromnonamers and top views. For purified CsgE a total of 2,452 particleswere analysed. Three-dimensional models were obtained as described forthe CsgG-CsgE cryo-EM analysis below and refined by several rounds ofmulti-reference alignment (MRA), multi-statistical analysis (MSA) andanchor set refinement. In all cases, after normalization and centring,images were classified using IMAGIC-4D as described in the cryo-EMsection. The best classes corresponding to characteristic views wereselected for each set of particles. Symmetry determination of CsgG topviews was performed using the best class averages with roughly 20 imagesper class. The rotational autocorrelation function was calculated usingIMAGIC and plotted.

Example 9: Negative Stain EM and Symmetry Determination

Negative stain EM was used to monitor in-solution oligomerization statesof CsgG, CsgGC1S and CsgE. CsgE, CsgGC1S and amphipol-bound CsgG wereadjusted to a concentration of 0.05 mg ml-1 and applied toglow-discharged carbon-coated copper grids (CF-400; Electron MicroscopySciences). After 1 min incubation, samples were blotted, then washed andstained in 2% uranyl acetate. Images were collected on a Tecnai T12BioTWIN LaB6 microscope operating at a voltage of 120 kV, at amagnification of ×49,000 and defocus between 800 and 2,000 nm. Contrasttransfer function (CTF), phase flipping and particle selection wereperformed as described for cryo-EM. For membrane-extracted CsgG,octadecameric particles (1,780 in all) were analysed separately fromnonamers and top views. For purified CsgE a total of 2,452 particleswere analysed. Three-dimensional models were obtained as described forthe CsgG-CsgE cryo-EM analysis below and refined by several rounds ofmulti-reference alignment (MRA), multi-statistical analysis (MSA) andanchor set refinement. In all cases, after normalization and centring,images were classified using IMAGIC-4D as described in the cryo-EMsection. The best classes corresponding to characteristic views wereselected for each set of particles. Symmetry determination of CsgG topviews was performed using the best class averages with roughly 20 imagesper class. The rotational autocorrelation function was calculated usingIMAGIC and plotted.

Example 10: CsgG-CsgE Complex Formation

For CsgG-CsgE complex formation, the solubilizing detergents in purifiedCsgG were exchanged for Amphipols A8-35 (Anatrace) by adding 120 ml ofCsgG (24 mg ml⁻¹ protein in 0.5% C8E4, 4 mM LDAO, 25 mM Tris-HCl pH 8.0,500 mM NaCl, 1 mM DTT) to 300 ml of detergent-destabilized liposomes (1mg ml⁻¹ 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 0.4%LDAO) and incubating for 5 min on ice before the addition of 90 ml ofA8-35 amphipols at 100 mg ml⁻¹ stock. After an additional 15 minincubation on ice, the sample was loaded on a Superose 6 10/300 GL (GEHealthcare) column and gel filtration was performed in 200 mM NaCl, 2.5%xylitol, 25 mM Tris-HCl pH 8, 0.2 mM DTT. An equal volume of purifiedmonomeric CsgE in 200 mM NaCl, 2.5% xylitol, 25 mM Tris-HCl pH 8, 0.2 mMDTT was added to the amphipol-solubilized CsgG at final proteinconcentrations of 15 and 5 mM for CsgE and CsgG, respectively, and thesample was run at 125 V at 18° C. on a 4.5% native PAGE in 0.5×TBEbuffer. For the second, denaturing dimension, the band corresponding tothe CsgG-CsgE complex was cut out of unstained lanes run in parallel onthe same gel, boiled for 5 min in Laemmli buffer (60 mM Tris-HCl pH 6.8,2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) andrun on 4-20% SDS-PAGE. Purified CsgE and CsgG were run alongside thecomplex as control samples. Gels were stained with InstantBlue Coomassiefor visual inspection or SYPRO orange for stoichiometry assessment ofthe CsgG-CsgE complex by fluorescence detection (Typhoon FLA 9000) ofthe CsgE and CsgG bands on SDS-PAGE, yielding a CsgG/CsgE ratio of 0.97.

Example 11: CsgG-CsgE Cryo-EM

Cryo-electron microscopy was used to determine the in-solution structureof the C₉ CsgG-CsgE complex. CsgG-CsgE complex prepared as describedabove was bound and eluted from a HisTrap FF (GE Healthcare) to removeunbound CsgG, and on elution it was immediately applied to QuantifoilR2/2 carbon coated grids (Quantifoil Micro Tools GmbH) that had beenglow-discharged at 20 mA for 30 s. Samples were plunge-frozen in liquidnitrogen using an automated system (Leica) and observed under a FEI F20microscope operating at a voltage of 200 kV, a nominal magnification of×50,000 under low-dose conditions and a defocus range of 1.4-3 mm. Imageframes were recorded on a Falcon II detector. The pixel size at thespecimen level was 1.9 Å per pixel. The CTF parameters were assessedusing CTFFIND3 (Mindell, J. A. & Grigorieff, N., J. Struct. Biol. 2003,142, 334-347), and the phase flipping was done in SPIDER (Shaikh, T. R.et al., Nature Protocols, 2008, 3, 1941-1974). Particles wereautomatically selected from CTF-corrected micrographs using BOXER(EMAN2; Tang, G. et al., J. Struct. Biol., 2007, 157, 38-46). Imageswith an astigmatism of more than 10% were discarded. A total of 1,221particles were selected from 75 micrographs and windowed into128-pixel×128-pixel boxes. Images were normalized to the same mean andstandard deviation and high-pass filtered at a low-resolution cut-off of˜200 Å. They were centred and then subjected to a first round of MSA. Aninitial reference set was obtained using reference free classificationin IMAGIC-4D (Image Science Software). The best classes corresponding tocharacteristic side views of the C₉ cylindrical particles were used asreferences for the MRA. For CsgG-CsgE complex, the firstthree-dimensional model was calculated from the best 125 characteristicviews (with good contrast and well-defined features) encompassing 1,221particles of the complex with orientations determined by angularreconstitution (Image Science Software). The three-dimensional map wasrefined by iterative rounds of MRA, MSA and anchor set refinement. Theresolution was estimated to be 24 Å by Fourier shell correlation (FSC)according to the 0.5 criteria level (FIG. 5).Visualization of the map and Figures was performed in UCSF Chimera(Pettersen, E. F. et al., J. Comput. Chem., 2004, 25, 1605-1612).

Example 12: Single-Channel Current Analysis of CsgG and CsgG+CsgE Pores

Under negative field potential, CsgG pores show two conductance states.FIG. 5 shows representative single-channel current traces of,respectively, the normal (measured at +50, 0 and −50 mV) and thelow-conductance forms (measured at 0, +50 and −50 mV). No conversionsbetween both states were observed during the total observation time(n=22), indicating that the conductance states have long lifetimes(second to minute timescale). The lower left panel shows a currenthistogram for the normal and low-conductance forms of CsgG poresacquired at +50 and −50 mV (n=33). I-V curves for CsgG pores withregular and low conductance are shown in the lower right panel. The datarepresent averages and standard deviations from at least fourindependent recordings. The nature or physiological existence of thelow-conductance form is unknown.FIG. 6 shows the results of electrophysiology of CsgG channels titratedwith the accessory factor CsgE. The plots display the fraction of open,intermediate and closed channels as a function of CsgE concentration.Open and closed states of CsgG are illustrated in FIG. 6 (0 nM and 100nM CsgE respectively). Increasing the concentration of CsgE to more than10 nM leads to the closure of CsgG pores. The effect occurs at +50 mV(left) and −50 mV (right), ruling out the possibility that the poreblockade is caused by electrophoresis of CsgE (calculated pl 4.7) intothe CsgG pore. An infrequent (<5%) intermediate state has roughly halfthe conductance of the open channel. It may represent CsgE-inducedincomplete closures of the CsgG channel; alternatively, it couldrepresent the temporary formation of a CsgG dimer caused by the bindingof residual CsgG monomer from the electrolyte solution to themembrane-embedded pore. The fraction for the three states was obtainedfrom all-point histogram analysis of single-channel current traces. Thehistograms yielded peak areas for up to three states, and the fractionfor a given state was obtained by dividing the corresponding peak areaby the sum of all other states in the recording. Under negative fieldpotential, two open conductance states are discerned, similar to theobservations for CsgG (see a). Because both open channel variations wereblocked by higher CsgE concentrations, the ‘open’ traces in FIG. 6 (0nM) combine both conductance forms. The data in the plot representaverages and standard deviations from three independent recordings.The crystal structure, size-exclusion chromatography and EM show thatdetergent extracted CsgG pores form non-native tail-to-tail stackeddimers (for example, two nonamers as D9 particle, FIG. 7) at higherprotein concentration. These dimers can also be observed insingle-channel recordings. The upper panel shows the single-channelcurrent trace of a stacked CsgG pore at +50, 0 and −50 mV (left toright). The lower left panel shows a current histogram of dimeric CsgGpores recorded at +50 and −50 mV. The experimental conductances of+16.2±1.8 and −16.0±3.0 pA (n=15) at +50 and −50 mV, respectively, arenear the theoretically calculated value of 23 pA. The lower right panelshows an I-V curve for the stacked CsgG pores. The data representaverages and standard deviations from six independent recordings.The ability of CsgE to bind and block stacked CsgG pores was tested byelectrophysiology. Shown are single-channel current traces of stackedCsgG pore in the presence of 10 or 100 nM CsgE at +50 mV (upper) and −50mV (lower) are shown. The current traces indicate that otherwisesaturating concentrations of CsgE do not lead to pore closure forstacked CsgG dimers. These observations are in good agreement with themapping of the CsgG-CsgE contact zone to helix 2 and the mouth of theCsgG periplasmic cavity as discerned by EM and site-directed mutagenesis(FIGS. 5 and 6).

Example 13: Bile Salt Toxicity Assay

Outer-membrane permeability was investigated by decreased growth on agarplates containing bile salts. Tenfold serial dilutions of E. coli LSR12(Chapman, M. R. et al., Science, 2002, 295, 851-855) cells (5 ml)harbouring both pLR42 (Nenninger, A. A. et al., Mol. Microbiol., 2011,81, 486-499) and pMC2 (Robinson, L. S. et al., Mol. Microbiol., 2006,59, 870-881) (or derived helix 2 mutants) were spotted on McConkey agarplates containing 100 mg l⁻¹ ampicillin, 25 mg l⁻¹ chloramphenicol, 1 mMIPTG with or without 0.2% (w/v) L-arabinose. After incubation overnightat 37° C., colony growth was examined.

Example 14: Single-Channel Current Recordings

Single-channel current recordings were performed using parallelhigh-resolution electrical recording with the Orbit 16 kit from Nanion.In brief, horizontal bilayers of1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids) wereformed over microcavities (of subpicolitre volume) in a 16-channelmultielectrode cavity array (MECA) chip (Ionera) (del Rio Martinez, J.M., ACS NanoSmall, 2014, 5, 8080-8088). Both the cis and trans cavitiesabove and below the bilayer contained 1.0 M KCl, 25 mM Tris-HCl pH 8.0.To insert channels into the membrane, CsgG dissolved in 25 mM Tris-HClpH 8.0, 500 mM NaCl, 1 mM DTT, 0.5% C8E4, 5 mM LDAO was added to the ciscompartment to a final concentration of 90-300 nM. To test theinteraction of the CsgG channel with CsgE, a solution of the latterprotein dissolved in 25 mM Tris-HCl pH 8.0, 150 mM NaCl was added to thecis compartment to final concentrations of 0.1, 1, 10 and 100 nM.Transmembrane currents were recorded at a holding potential of +50 mVand −50 mV (with the cis side grounded) using a Tecella Triton16-channel amplifier at a low-pass filtering frequency of 3 kHz and asampling frequency of 10 kHz. Current traces were analysed using theClampfit of the pClamp suite (Molecular Devices). Plots were generatedusing Origin 8.6 (Microcal) (Movileanu, L., Nature Biotechnol., 2000,18, 1091-1095).Measured currents were compared with those calculated based on the poredimensions of the CsgG X-ray structure, modelled to be composed of threesegments: the transmembrane section, the periplasmic vestibule, and theinner channel constriction connecting the two. The first two segmentswere modeled to be of conical shape while the constriction wasrepresented as a cylinder. The corresponding resistances R₁, R₂ and R₃,respectively, were calculated as

R ₁ =L ₁/(πD ₁ d ₁κ)

R ₂ =L ₂/(πD ₂ d ₂κ)

R ₃ =L ₃/(πd ₁ d ₂κ)

where L₁, L₂ and L₃ are the axial lengths of the segments, measuring3.5, 4.0 and 2.0 nm, respectively, and D₁, d₁, D₂ and d₂ are the maximumand minimum diameters of segments 1 and 2, measuring 4.0, 0.8, 3.4 and0.8 nm, respectively. The conductivity κ has a value of 10.6 S m⁻¹. Thecurrent was calculated by inserting R₁, R₂ and R₃ and voltage V=50 mVinto

I=V/(R ₁ +R ₂ +R ₃)

Access resistance was not found to alter the predicted currentsignificantly.Single channel current recordings such as those described above may bemade in the presence of analytes, thereby allowing the channel to assumethe role of a biosensor.

Example 15: Molecular Dynamics Simulation of CsgG Constriction withModel Polyalanine Chain

The CsgG constriction has been modelled with a polyalanine chainthreaded through the channel in an extended conformation, shown in FIG.9 in a C-terminal to N-terminal direction. Substrate passage through theCsgG transporter is itself not sequence specific (Nenninger, A. A. etal., Mol. Microbiol., 2011, 81, 486-499; Van Gerven, N. et al., Mol.Microbiol. 2014, 91, 1022-1035 2014). For clarity, a polyalanine chainwas used for modeling the putative interactions of a passing polypeptidechain. The modelled area is composed of nine concentric CsgG C-loops,each comprising residues 47-58. Side chains lining the constriction areshown in stick representation with Asn 55 and Phe 56 marked. Solventmolecules (water) within 10 Å of the polyalanine residues inside theconstriction (residues labelled +1 to +5) are shown as dots. FIG. 9cshows the modelled solvation of the polyalanine chain, positioned asshown in FIG. 9b and with C-loops removed for clarity (shown solventmolecules are those within 10 Å of the full polyalanine chain). At theheight of ring of Asn 55 and Phe 56, the solvation of the polyalaninechain is reduced to a single water shell that bridges the peptidebackbone and amide-clamp side chains. Most side chains in the Tyr 51ring have rotated towards the solvent in comparison with their inward,centre-pointing position observed in the CsgG (and the CsgG_(C1S)) X-raystructure. The model is the result of a 40 ns all-atom explicit solventmolecular dynamics simulation with GROMACS (Pronk, S. et al.,Bioinformatics, 2013, 29, 845-854) using the AMBER99SB-ILDN(Lindorff-Larsen, K. et al., Proteins 2010, 78, 1950-1958) force fieldand with the Ca atoms of the residues at the extremity of the C-loop(Gln 47 and Thr 58) positionally restricted.

Example 16: Use of the CsgG Nanopore for Nucleic Acid Sequencing

The Phi29 DNA polymerase (DNAP) may be used as a molecular motor with amutant or wild type CsgG nanopore located within a membrane to allowcontrolled movement of an oligomeric probe DNA strand through the pore.A voltage may be applied across the pore and a current generated fromthe movement of ions in a salt solution on either side of the nanopore.As the probe DNA moves through the pore, the ionic flow through the porechanges with respect to the DNA. This information has been shown to besequence dependent and allows for the sequence of the probe to be readwith accuracy from current measurements such as those described above inExample 14.

Example 17

This Example describes the simulations which were run to investigate DNAbehaviour within CsgG.

Materials and Methods

Steered molecular dynamics simulations were performed to investigate themagnitude of the energetic barrier of CsgG-Eco and various mutants toDNA translocation. Simulations were performed using the GROMACS packageversion 4.0.5, with the GROMOS 53a6 forcefield and the SPC water model.The structure of CsgG-Eco (SEQ ID NO: 390) was taken from the proteindata bank, accession code 4UV3. In order to make models of the CsgG-Ecomutants, the wild-type protein structure was mutated using PyMOL. Themutants studied were CsgG-Eco-(F56A) (SEQ ID NO: 390 with mutationF56A), CsgG-Eco-(F56A-N55S) (SEQ ID NO: 390 with mutations F56A/N55S)and CsgG-Eco-(F56A-N55S-Y51A) (SEQ ID NO: 390 with mutationsF56A/N55S/Y51A).

DNA was then placed into the pores. Two different systems were set up:

-   -   i. A single guanine nucleotide was placed into the pore, just        above the constriction region (approximately 5-10 Angstroms        above the residue 56 ring)    -   ii. A single strand of DNA (ssDNA) was placed along the pore        axis, with the 5′ end towards the beta-barrel side of the pore.        In this set up, the ssDNA was pre-threaded through the entire        length of the pore.

The simulation box was then solvated and then energy minimised using thesteepest descents algorithm.

Each system was simulated in the NPT ensemble, using the Berendsenthermostat and Berendsen barostat to 300 K. Throughout the simulation,restraints were applied to the backbone of the pore.

In order to pull the DNA through the pore, a pulling force was appliedto the phosphorus atom in the single guanine simulations. In the ssDNAsimulations the pulling force was applied to the phosphorus atom at the5′ end of the strand. The puling force was applied at a constantvelocity by connecting a spring between the DNA phosphorus atommentioned above and an imaginary point travelling at a constant velocityparallel to the pore axis. Note that the spring does not have any shapenor does it undergo any hydrodynamic drag. The spring constant was equalto 5 kJmol⁻¹ Å⁻².

Results Single G Translocation

As shown in FIG. 12, a plot of the pulling force versus time shows thatthere is a large barrier for nucleotide entry into the ring ofphenylalanine residues F56 in the wild type CsgG-Eco pore. There was nosignificant barrier to guanine translocation observed for the CsgG-Ecomutants studied.

ssDNA Translocation

For ssDNA translocation, two simulations were run per pore with each runhaving a different applied pulling velocity (100 Å/ns and 10 Å/ns). Asshown in FIG. 13, which illustrates the faster puling velocitysimulations, the CsgG wild-type pore required the largest pulling forceto enable ssDNA translocation. As shown in FIG. 14, which illustratesthe slower pulling velocity simulations, both the CsgG-Eco (wild-type,SEQ ID NO: 390) and CsgG-Eco-(F56A) pores required the largest appliedforce to enable ssDNA translocation. Comparisons between the pullingforce required for ssDNA translocation through CsgG and MspA baselinepore, suggest that mutation of the CsgG pore is required to allow asimilar level of ssDNA translocation.

Example 18

This Example describes the characterisation of several CsgG mutants.

Materials and Methods

Prior to setting up the experiment, DNA construct X (final concentration0.1 nM, see FIG. 22 for cartoon representation of construct X anddescription) was pre-incubated at room temperature for five minutes withT4 Dda-E94C/C109A/C136A/A360C (SEQ ID NO: 412 with mutationsE94C/C109A/C136A/A360C, final concentration added to the nanopore system10 nM, which was provided in buffer (151.5 mM KCl, 25 mM potassiumphosphate, 5% glycerol, pH 7.0, 1 mM EDTA)). After five minutes, TMAD(100 μM) was added to the pre-mix and the mixture incubated for afurther 5 minutes. Finally, MgCl2 (1.5 mM final concentration added tothe nanopore system), ATP (1.5 mM final concentration added to thenanopore system), KCl (500 mM final concentration added to the nanoporesystem) and potassium phosphate buffer (25 mM final concentration addedto the nanopore system) were added to the pre-mix.

Electrical measurements were acquired from a variety of single CsgGnanopores inserted in block co-polymer in buffer (25 mM K Phosphatebuffer, 150 mM Potassium Ferrocyanide (II), 150 mM PotassiumFerricyanide (III), pH 8.0). After achieving a single pore inserted inthe block co-polymer, then buffer (2 mL, 25 mM K Phosphate buffer, 150mM Potassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH8.0) was flowed through the system to remove any excess CsgG nanopores.150 uL of 500 mM KCl, 25 mM K Phosphate, 1.5 mM MgCl2, 1.5 mM ATP, pH8.0was then flowed through the system. After 10 minutes a 150 uL of 500 mMKCl, 25 mM potassium phosphate, 1.5 mM MgCl2, 1.5 mM ATP, pH8.0 wasflowed through the system and then the enzyme (T4Dda-E94C/C109A/C136A/A360C, 10 nM final concentration), DNA construct X(0.1 nM final concentration), fuel (MgCl2 1.5 mM final concentration,ATP 1.5 mM final concentration) premix (150 μL total) was then flowedinto the single nanopore experimental system. The experiment was run at−120 mV and helicase-controlled DNA movement monitored.

Results Pores Showing Increased Range (FIGS. 15 to 17, and 27 to 39)

CsgG-Eco-(StrepII(C)) (SEQ ID NO: 390 where StepII(C) is SEQ ID NO: 435and is attached at the C-terminus) has a range of ˜10 pA (see FIG.15(a)) whereas the CsgG-Eco pore mutants below exhibited an increasedcurrent range—1—CsgG-Eco-(Y51N-F56A-D149N-E185R-E201N-E203N-StrepII(C))9 (SEQ ID NO:390 with mutations Y51N/F56A/D149N/E185R/E201N/E203N where StepII(C) isSEQ ID NO: 435 and is attached at the C-terminus) exhibited a range of˜30 pA (See FIG. 15(b)).2—CsgG-Eco-(N55A-StrepII(C))9 (SEQ ID NO: 390 with mutation N55A whereStepII(C) is has SEQ ID NO: 435 and is attached at the C-terminus)exhibited a range of ˜35 pA (see FIG. 15(c)).3—CsgG-Eco-(N55S-StrepII(C))9 (SEQ ID NO: 390 with mutations N55S whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus) exhibiteda range of ˜40 pA (see FIG. 16(a)).4—CsgG-Eco-(Y51N-StrepII(C))9 (SEQ ID NO: 390 with mutation Y51N whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus) exhibiteda range of ˜40 pA (see FIG. 16(b)).5—CsgG-Eco-(Y51A-F56A-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51A/F56A where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜30 pA (see FIG. 16(c)).6—CsgG-Eco-(Y51A-F56N-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51A/F56N where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜20 pA (see FIG. 17(a)).7—CsgG-Eco-(Y51A-N55S-F56A-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51A/N55S/F56A where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜30 pA (see FIG. 17(b)).8—CsgG-Eco-(Y51A-N55S-F56N-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51A/N55S/F56N where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜30 pA (see FIG. 17(c)).13—CsgG-Eco-(F56H-StrepII(C))9 (SEQ ID NO: 390 with mutation F56H whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus) exhibiteda range of ˜35 pA (see FIG. 27).14—CsgG-Eco-(F56Q-StrepII(C))9 (SEQ ID NO: 390 with mutation F56Q whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus) exhibiteda range of ˜40 pA (see FIG. 28).15—CsgG-Eco-(F56T-StrepII(C))9 (SEQ ID NO: 390 with mutation F56T whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus) exhibiteda range of ˜35 pA (see FIG. 29).16—CsgG-Eco-(S54P/F56A-StrepII(C))9 (SEQ ID NO: 390 with mutationS54P/F56A where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜35 pA (see FIG. 30).17—CsgG-Eco-(Y51T/F56A-StrepII(C))9 (SEQ ID NO: 390 with mutationY51T/F56A where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜30 pA (see FIG. 31).18—CsgG-Eco-(F56P-StrepII(C))9 (SEQ ID NO: 390 with mutation F56P whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus) exhibiteda range of ˜30 pA (see FIG. 32).19—CsgG-Eco-(F6A-StrepII(C))9 (SEQ ID NO: 390 with mutation F56A whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus) exhibiteda range of ˜40 pA (see FIG. 33).20—CsgG-Eco-(Y51T/F56Q-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51T/F56Q where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜30 pA (see FIG. 34).21—CsgG-Eco-(N55S/F56Q-StrepII(C))9 (SEQ ID NO: 390 with mutationsN55S/F56Q where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜35 pA (see FIG. 35).22—CsgG-Eco-(Y51T/N55S/F56Q-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51T/N55S/F56Q where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜35 pA (see FIG. 36).23—CsgG-Eco-(F56Q/N102R-StrepII(C))9 (SEQ ID NO: 390 with mutationsF56Q/N102R where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜30 pA (see FIG. 37).24—CsgG-Eco-(Y51Q/F56Q-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51Q/F56Q where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜40 pA (see FIG. 38).25—CsgG-Eco-(Y51A/F56Q-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51A/F56Q where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus) exhibited a range of ˜35 pA (see FIG. 39).

Pores Showing Increased Throughput (FIGS. 18 and 19)

As can be seen from FIGS. 18 and 19, the following mutant pores (9-12below) exhibited multiple helicase controlled DNA movements (Labelled asX in FIGS. 18 and 19) per channel in 4 hours, whereasCsgG-Eco-(StrepII(C)) (SEQ ID NO: 390 where StepII(C) is SEQ ID NO: 435and is attached at the C-terminus) shown in FIG. 18(a) frequentlyexhibited only 1 or 2 helicase controlled DNA movements (labelled as Xin FIG. 18(a)) per channel in 4 hours and instead exhibited prolongedblock regions (labeled as Y in FIG. 18(a)).

9—CsgG-Eco-(D149N-E185N-E203N-StrepII(C))9 (SEQ ID NO: 390 withmutations D149N/E185N/E203N where StepII(C) is SEQ ID NO: 435 and isattached at the C-terminus) (FIG. 18(b))10—CsgG-Eco-(D149N-E185N-E201N-E203N-StrepII(C))9 (SEQ ID NO: 390 withmutations D149N/E185N/E201N/E203N where StepII(C) is SEQ ID NO: 435 andis attached at the C-terminus) (FIG. 18(c))11—CsgG-Eco-(D149N-E185R-D195N-E201N-E203N)-StrepII(C))9 (SEQ ID NO: 390with mutations D149N/E185R/D195N/E201N/E203N where StepII(C) is SEQ IDNO: 435 and is attached at the C-terminus) (FIG. 19(a))12—CsgG-Eco-(D149N-E185R-D195N-E201R-E203N)-StrepII(C))9 (SEQ ID NO: 390with mutations D149N/E185R/D195N/E201R/E203N where StepII(C) is SEQ IDNO: 435 and is attached at the C-terminus) (FIG. 19(b))

Pore Showing Increased Insertion (FIGS. 20 and 21)

As can be seen by comparing FIGS. 20 and 21, the mutant poreCsgG-Eco-(T150I-StrepII(C))9 (SEQ ID NO: 390 with mutations T150I whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus) shown inFIG. 21. was present in the membrane in increased pore numbers (˜4-5fold) compared with the CsgG-Eco-(StrepII(C)) (SEQ ID NO: 390 whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus) pore(shown in FIG. 20). Arrows in FIGS. 20 and 21 illustrated the number ofCsgG-Eco nanopores which inserted into the block co-polymer in a 4 hourexperiment (130-140 in FIG. 20 and 1-11 in FIG. 21 each corresponded toa separate nanopore experiment). For CsgG-Eco-(StrepII(C)) (SEQ ID NO:390 where StepII(C) is SEQ ID NO: 435 and is attached at the C-terminus)three experiments showed insertion of one nanopore, whereas for themutant pore (CsgG-Eco-(T150I-StrepII(C))9) each experiment showedinsertion of at least one nanopore and several experiments showedmultiple pore insertions.

Example 19

This example described an E. coli purification method developed topurify the CsgG pore.

Materials and Methods

DNA encoding the polypeptide Pro-CsgG-Eco-(StrepII(C)) (SEQ ID NO: 390where StepII(C) is SEQ ID NO: 435 and is attached at the C-terminus andwhere Pro is SEQ ID NO: 436 and is attached at the N-terminus) wassynthesised in GenScript USA Inc. and cloned into a pT7 vectorcontaining ampicillin resistance gene. Protein expression of the pT7vector was induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG). Theconcentration of the DNA solution was adjusted to 400 ng/uL. DNA (1 μl)was used to transform Lemo21(DE3) competent E. coli cells (50 μl, NEB,catalogue number C2528H). Prior to transformation, the CsgG gene wasknocked out from Lemo21(DE3) cells (Gene Bridges GmbH, Germany). Thecells were then plated out on LB agar containing ampicillin (0.1 mg/mL)and incubated for approx 16 hours at 37° C.

Bacterial colonies grown on LB plates, containing ampicillin,incorporated the CsgG plasmid. One such colony was used to inoculate astarter culture of LB media (100 mL) containing carbenicillin (0.1mg/mL). The starter culture was grown at 37° C. with agitation untilOD600 was reached to 1.0-1.2. The starter culture was used to inoculatea fresh 500 mL of LB media containing carbenicillin (0.1 mg/mL) andRhamnose (500 μM) to an O.D. 600 of 0.1. The culture was grown at 37° C.with agitation until OD600 reached 0.6. The temperature of the culturewas then adjusted to 18° C. and induction was initiated by the additionof IPTG (0.2 mM final concentration). Induction was carried out forapproximately 18 hours with agitation at 18° C.

Following induction, the culture was pelleted by centrifugation at 6,000g for 30 minutes. The pellet was resuspended in 50 mM Tris, 300 mM NaCl,containing protease inhibitors (Merck Millipore 539138), benzonasenuclease (Sigma E1014) and 1× bugbuster (Merck Millipore 70921) pH8.0(approximately 10 mL of buffer per gram of pellet). Suspension was mixedwell until it was fully homogeneous, the sample was then transferred toroller mixer at 4° C. for approx 5 hours. Lysate was pelleted bycentrifugation at 20,000 g for 45 minutes and the supernatant wasfiltered through 0.22 μM PES syringe filter. Supernatant which containedCsgG (known as sample 1) was taken forward for purification by columnchromatography.

Sample 1 was applied to a 5 mL Strep Trap column (GE Healthcare). Thecolumn was washed with 25 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.01% DDM pH8until a stable baseline of 10 column volumes was maintained. The columnwas then washed with 25 mM Tris, 2M NaCl, 2 mM EDTA, 0.01% DDM pH8before being returned to the 150 mM buffer. Elution was carried out with10 mM desthiobiotin. An example of a chromatography trace of Strep trap(GE Healthcare) purification of a CsgG protein is shown in FIG. 23. Theelution peak is labelled E1. FIG. 24 shows an example of a typicalSDS-PAGE visualization of CsgG-Eco protein after the initial Streppurification. Lanes 1-3 shows the main elution peak (labelled E1 in FIG.23) which contained CsgG protein as indicated by the arrow. Lanes 4-6corresponded to elution fractions of the tall of the main elution peak(labelled E1 in FIG. 23) which contained contaminants.

The elution peak was pooled and heated to 65° C. for 15 minutes toremove heat unstable contaminated proteins. The heated solution wassubjected to centrifugation at 20,000 g for 10 minutes and the pelletwas discarded. The supernatant was subjected to gel filtration on a 120mL Sephadex S200 column (GE Healthcare) in 25 mM Tris, 150 mM NaCl, 2 mMEDTA, 0.01% DDM, 0.1% SDS pH8. Monitoring was carried out at 220 nM dueto low Tryptophan component of protein. The sample was eluted atapproximately 55 mL volume (FIG. 25 shows the size exclusion columntrace with the 55 mL sample peak labeled with a star). The elution peakwas run on a 4-20% TGX (see FIG. 26, Bio Rad) to confirm the presence ofthe pore of interest CsgG-Eco-(StrepII(C)) (SEQ ID NO: 390 whereStepII(C) is SEQ ID NO: 435 and is attached at the C-terminus).Identified fractions were pooled and concentrated by 50 kD Amicon spincolumn.

Example 20

This example describes the simulations which were run to investigate theinteraction between CsgG-Eco-(Y51T/F56Q)-StrepII(C))9 (SEQ ID NO: 390with mutations Y51T/F56Q where StepII(C) is SEQ ID NO: 435 and isattached at the C-terminus pore mutant No. 20) with T4Dda-(E94C/C109A/C136A/A360C) (SEQ ID NO: 412 with mutationsE94C/C109A/C136A/A360C and then (ΔM1)G1G2).

Simulation Methods

Simulations were performed using the GROMACS package version 4.0.5, withthe GROMOS 53a6 forcefield and the SPC water model.The CsgG-Eco-(Y51T/F56Q)-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51T/F56Q where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus pore mutant No. 20) model was based on the crystal structuresof CsgG found in the protein data bank, accession codes 4UV3 and 4Q79.The relevant mutations were made using PyMOL. The resultant pore modelwas then energy minimised using the steepest descents algorithm. The T4Oda-(E94C/C109A/C136A/A360C) (SEQ ID NO: 412 with mutationsE94C/C109A/C136N/A360C and then (ΔM1)G1G2) model was based on theDda1993 structure found in the protein data bank, accession code 3UPU.Again, relevant mutations were made using PyMOL, and the model wasenergy minimised using the steepest descents algorithm.The T4 Dda-(E94C/C109A/C136A/A360C) (SEQ ID NO: 412 with mutationsE94C/C109A/C136A/A360C and then (ΔM1)G1G2) model was then placed aboveCsgG-Eco-(Y51T/F56Q)-StrepII(C))9 (SEQ ID NO: 390 with mutationsY51T/F56Q where StepII(C) is SEQ ID NO: 435 and is attached at theC-terminus pore mutant No. 20). Three simulations were performed with adifferent initial enzyme conformation (Runs 1 to 3 (0 ns), see FIG. 40):In all enzyme conformations, the enzyme was oriented such that the 5′end of the DNA was pointing towards the pore, and the enzyme wasunrestrained throughout the simulation. The pore backbone was restrainedand the simulation box was solvated. The system was simulated in the NPTensemble for 40 ns, using the Berendsen thermostat and Berendsenbarostat to 300 K.The contacts between the enzyme and pore were analysed using bothGROMACS analysis software and also locally written code. The tablesbelow show the number of contacts observed for both pore and enzymeamino acids. Tables 6-8 show the amino acid contact points on pore whichinteract with the amino acid contact points on the enzyme. In two out ofthe three simulations the enzyme tilts on top of the pore (see run 2 and3 (20, 30 and 40 ns), FIGS. 40 and 41). Run 1 shows that the enzyme hasnot tilted and so points that are shown to have high interaction intable 6 can be optimised in order to increase enzyme stability on thepore cap.

Table 6 = run 1 enzyme and pore contact interactions Pore Enzyme #contacts ASN 102 ASP 198 8200 ASN 102 TYR 438 8130 GLN 100 ASP 212 7369GLU 101 TRP 195 5979 ARG 97 TYR 350 4873 GLU 101 LEU 215 4851 ASN 102TRP 195 3988 ARG 97 TYR 415 3798 GLU 101 TYR 350 3759 LEU 113 ASP 2123718 ASN 102 LYS 358 3124 ARG 97 GLY 211 2765 GLU 101 CYS 412 2715 ARG97 GLY 193 2708 ASN 102 ILE 196 2342 GLU 101 TYR 415 2268 GLU 101 ARG216 2158 ARG 110 THR 213 2094 ARG 110 ASP 212 2066 GLY 103 ARG 216 1456GLU 101 TYR 318 1333 ASN 102 GLU 347 1316 GLU 101 LYS 194 1310 ARG 97PRO 411 1203 GLU 101 LYS 358 1161 ASN 102 ARG 216 1132 ARG 97 TRP 195888 LYS 94 TYR 415 793 ASN 102 PRO 315 696 ASN 102 LYS 247 541 GLU 101ALA 214 449 ASN 102 ASP 346 440 ARG 97 ALA 214 366 ARG 97 LYS 194 336GLU 101 ASP 212 302 ARG 97 VAL 439 267 ARG 110 THR 210 263 ARG 97 THR210 259 ARG 97 GLN 422 257 GLU 101 TYR 409 228 ALA 98 TRP 195 207 GLU101 LYS 247 201 ASN 102 GLU 317 179 ARG 110 ARG 216 147 ARG 97 ASP 212108 ASN 102 VAL 314 87 GLU 101 THR 213 72 ASN 102 LYS 255 70 VAL 105 ARG216 69 ASN 102 LEU 215 59 ASN 102 THR 210 55 ILE 111 ASP 212 48 ARG 97HIS 414 48 THR 104 ARG 216 36 ASN 102 TYR 197 32 GLN 100 THR 213 30 ASN102 GLU 361 28 ARG 97 VAL 418 28 ALA 98 TYR 415 27 GLU 101 LEU 354 17GLU 101 TYR 197 16 ASN 102 GLY 316 16 ARG 97 GLU 361 16 ARG 97 GLU 34714 ILE 107 ARG 216 12 ASN 102 GLY 208 12 ARG 97 TYR 409 11 ARG 97 LYS247 11 GLU 101 LYS 364 8 ARG 97 PHE 209 7 LYS 94 GLU 419 6 GLU 101 PRO411 5 GLU 101 GLU 317 5 ASN 102 ILE 251 5 ARG 97 LEU 354 5 LYS 94 VAL418 3 ASN 102 ARG 321 3 ARG 97 LYS 243 3 LYS 94 CYS 412 2 LEU 113 THR210 2 GLY 103 GLU 317 2 GLU 101 LYS 351 2 ASN 102 TYR 318 2 ASN 102 MET219 2 ASN 102 LYS 194 2 ARG 97 VAL 314 2 ARG 97 LYS 364 2 THR 104 PRO315 1 GLY 103 THR 213 1 GLU 101 PRO 315 1

Table 7 = run 2 enzyme and pore contact interactions Pore Enzyme #contacts GLU 101 THR 210 14155 SER 115 ASP 202 9477 ARG 97 THR 210 9064ASN 102 VAL 200 5323 THR 104 ASP 202 4476 ASN 102 ASN 221 3422 GLU 101PHE 437 3171 ARG 97 ASP 217 2698 GLU 101 ARG 216 2198 ARG 97 GLY 2081730 GLU 101 LYS 199 1710 SER 115 SER 224 1440 ASN 102 LYS 199 1351 ASN102 ASP 212 1298 ASN 102 ARG 405 1219 GLU 101 ARG 207 1180 ASN 102 SER224 1150 ASN 102 LYS 255 1114 ARG 97 ASP 198 946 GLU 101 PHE 209 931 ARG97 THR 213 791 ARG 97 ARG 216 599 ASN 102 THR 210 589 GLN 114 ASP 202530 ASN 102 ASP 202 492 ARG 97 ASP 212 490 GLY 103 ARG 405 474 THR 104SER 224 451 GLU 101 LYS 255 429 ASN 102 ASP 198 405 ASN 102 PHE 209 400ASN 102 ARG 178 316 ARG 110 GLU 258 309 ASN 102 ASN 180 257 GLN 100 PHE223 256 GLU 101 TYR 197 220 GLN 114 SER 228 212 LEU 113 PHE 223 210 ASN102 ILE 225 204 GLN 114 LYS 227 194 GLU 101 GLY 211 189 GLU 101 ASP 212174 LEU 113 SER 224 159 LEU 113 GLY 203 145 ARG 97 VAL 220 134 GLU 101THR 213 133 THR 104 SER 228 125 ARG 97 TYR 197 123 LYS 94 ASP 212 118ASN 102 ARG 216 110 ASN 102 ASN 235 108 ASN 102 GLY 211 104 GLU 101 ARG405 79 GLN 114 SER 224 69 ASN 102 VAL 220 63 LEU 113 LYS 227 49 ASN 102VAL 201 42 ARG 97 PHE 209 42 GLU 101 ASN 180 40 ARG 97 TYR 438 38 ARG 97ARG 207 32 ASN 102 PHE 407 28 SER 115 ASN 221 23 ARG 110 HIS 204 22 GLU101 PHE 223 21 ARG 97 ASP 189 19 ARG 110 PHE 223 16 THR 104 ILE 225 13GLY 103 ASN 180 11 ARG 97 LYS 194 11 GLU 101 PHE 407 10 ARG 97 MET 219 9THR 104 ASN 235 8 ARG 110 ARG 405 8 ARG 97 TRP 195 7 ILE 111 PHE 223 6GLU 101 GLY 208 6 LEU 113 ASP 202 5 GLU 101 ARG 178 5 ASN 102 THR 213 5ALA 98 ARG 216 5 ASN 102 ASP 217 4 ARG 97 LYS 199 4 THR 104 LEU 229 3THR 104 ARG 405 3 GLU 101 VAL 201 3 GLU 101 MET 219 3 ARG 110 ASP 202 3ARG 110 ARG 207 2 THR 104 VAL 201 1 GLY 103 SER 224 1 GLY 103 LYS 255 1GLY 103 GLU 258 1 GLY 103 ASN 235 1 GLU 101 ASP 198 1 ASN 102 PHE 437 1ARG 97 PHE 437 1 ARG 110 LYS 227 1

Table 8 = run 3 enzyme and pore contact interactions Pore Enzyme #contacts ARG 97 THR 174 15557 GLN 100 ASP 5 10353 GLU 101 LYS 177 9238ARG 97 SER 179 6630 LEU 116 ASP 202 6545 GLU 101 TYR 434 6524 SER 115ASP 202 5693 GLU 101 HIS 204 5457 ARG 97 GLN 10 5106 ARG 93 ASP 202 4646ARG 93 GLU 8 4446 SER 115 LYS 11 4342 LEU 113 ASP 5 3871 ASN 102 SER 2243605 GLU 101 ASN 12 3344 GLU 101 GLN 10 3327 ARG 97 GLU 175 3096 GLU 101SER 224 3028 LEU 116 GLU 8 2936 LYS 94 ASP 185 2708 ARG 97 ASN 180 2700GLU 101 PHE 3 2500 THR 104 LYS 11 2352 SER 115 GLU 8 2323 ARG 93 ASN 1801912 ASN 102 LYS 177 1838 LYS 94 ASP 198 1828 ARG 110 ASP 5 1714 ALA 98GLY 203 1701 ASN 102 ASN 12 1695 GLU 101 TYR 169 1691 ARG 97 THR 7 1593ARG 110 ASP 4 1404 ARG 97 ASP 212 1381 ASN 102 HIS 204 1226 ASN 102 ASN15 1173 ARG 97 VAL 176 1096 ALA 98 HIS 204 998 ARG 97 ASP 202 875 ASN102 TYR 434 850 ALA 98 ASN 12 716 GLU 101 THR 213 702 GLU 101 ARG 178642 GLU 101 ASN 221 600 ASN 102 LYS 11 588 ARG 97 ASP 217 585 ARG 97 ARG207 537 GLU 101 ARG 207 525 ARG 97 PHE 437 511 GLU 101 ARG 216 510 ASN102 LYS 19 482 ARG 97 HIS 204 473 LEU 113 LYS 11 409 ARG 97 THR 213 358ARG 93 ASP 212 354 ARG 97 TYR 169 316 ARG 97 GLY 203 308 ARG 97 ASP 435300 GLN 87 LYS 199 249 THR 104 ASN 15 221 ARG 97 ALA 181 220 ASN 102 LYS227 198 LYS 94 ARG 178 184 ASN 102 GLU 8 183 LEU 113 LEU 6 182 ARG 93SER 179 179 LEU 90 ASN 180 172 LEU 90 ASP 202 144 ARG 97 ILE 225 138 GLU101 ASN 15 135 GLU 101 LYS 19 113 LYS 94 ASN 180 109 LYS 94 GLU 175 105ARG 93 THR 7 81 LYS 94 ARG 207 77 GLN 100 PHE 3 72 ASN 102 ARG 216 66ARG 97 LYS 177 62 GLU 101 THR 210 59 ARG 97 ARG 178 56 LYS 94 ASP 212 55ARG 97 GLU 172 53 GLU 101 VAL 176 51 ALA 98 ARG 207 49 ARG 110 PHE 3 48ALA 98 ASP 202 47 ARG 97 VAL 200 40 ALA 98 VAL 201 36 LYS 94 THR 210 35ILE 111 ASP 5 32 ARG 97 ARG 405 27 LEU 90 VAL 200 26 ARG 97 THR 210 26GLY 103 PHE 3 25 GLU 101 PHE 209 25 ARG 97 ARG 216 22 ASN 102 VAL 220 21LYS 94 GLY 211 19 ARG 97 PHE 209 17 GLU 101 LYS 227 15 GLN 114 LYS 11 15GLY 103 LYS 19 13 ARG 97 PHE 3 13 GLU 101 THR 2 12 GLU 101 ILE 225 12ARG 97 ILE 184 12 ALA 98 GLU 8 12 ALA 98 ARG 178 12 ASN 102 ILE 225 11LYS 94 LYS 199 10 GLU 101 ARG 433 8 ARG 97 ASN 221 8 LYS 94 VAL 200 7ASN 102 ASP 202 7 ASN 102 ASN 221 7 ARG 97 LEU 173 7 SER 115 HIS 204 6ASN 102 GLY 203 6 GLU 101 CVS 171 5 ARG 97 ASN 12 5 ASN 102 PHE 223 4ASN 102 LYS 166 4 ARG 97 GLY 211 4 ARG 97 GLN 170 4 GLU 101 ARG 405 3ASN 102 PHE 3 3 GLU 101 GLU 175 2 ARG 97 VAL 220 2 ARG 93 GLY 203 2 LYS94 THR 174 1 LEU 90 LYS 199 1 LEU 116 ASN 180 1 LEU 113 ASP 212 1 LEU113 ASP 202 1 GLY 103 ASN 15 1 GLU 101 THR 7 1 GLU 101 PHE 437 1 GLN 114ASP 202 1 ASN 102 ARG 405 1 ARG 97 TYR 434 1 ARG 97 PRO 182 1 ARG 97 GLY9 1 ARG 97 GLU 8 1 ALA 99 ASP 202 1

Example 21: Ability of the CsgG Nanopore to the Capture Nucleic Acids inthe Channel Constriction

The use of nanopores for nucleic acid sequencing requires the captureand threading of single stranded DNA by the nanopore. In this example,single channel current traces of a CsgG WT protein were followed inpresence of a DNA hairpin carrying a single-stranded DNA overhang. Theexample trace presented in FIG. 56 shows the current which alters inresponse to the potential measured at +50 mV or −50 mV intervals(indicated by arrows). The downward current blockades in the last +50 mVsegment represent the threading of the single-stranded hairpin end intoinner pore construction leading to an almost complete current blockade.Reversal of the electrical field to −50 mV results in theelectrophoretic unblocking of the pore. A new +50 mV episode resultsagain in DNA hairpin binding and pore blockage. On the +50 mV segments,unfolding of the hairpin structure can lead to the termination of thecurrent blockade indicated by the reversal of the current blockade. Thehairpin with the sequence 3′ GCGGGGAGCGTATTAGAGTTGGATCGGATGCAGCTGGCTACTGACGTCATGACGTCAGTAGCCAGCATGCATCCGATC-5′(SEQ ID NO: 441) was added to the cis side of the chamber at a finalconcentration of 10 nM.

Example 22. This Example Describes the Generation of a Mutant CsgG Corewith an Altered Inner Constriction

In this example, the stability and channel properties are demonstratedfor CsgG-ΔPYPA, a mutant CsgG pore where the sequence PYPA (residues50-53) in the constriction loop is replaced by GG. For this mutant, theconstriction motif from position 38 to 63 corresponds to SEQ ID NO: 354.This mutation foresees in the removal of Y51 from the pore constrictionand the shortening of the constriction loop in order to reducecomplexity in the pore reading head, i.e. the narrowest part of the porewhere the conductivity measured during sensing applications is mostsensitive to the nature of the substrate binding or threading the pore.Replacement of the PYPA sequence retains stability of the CsgG nonamerand results in a pore with increase conductivity as shown in FIG. 57.The CsgG-ΔPYPA pore mutant was analysed with single-channel currentrecordings using parallel high-resolution electrical recording with theOrbit 16 kit from Nanion (Munich, Germany). Briefly, horizontal bilayersof 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids)were formed over microcavities (of sub-picoliter volume) in a 16-channelmultielectrode cavity array (MECA) chip (Ionera, Freiburg, Germany) 51.Both the cis and trans cavities above and below the bilayer contained1.0 M KCl, 25 mM Tris-HCl, pH 8.0. To insert channels into the membrane,CsgG dissolved in 25 mM Tris pH 8.0, 500 mM NaCl, 1 mM DTT, 0.5% C8E4, 5mM LDAO was added to the cis compartment to a final concentration of 100nM. HTransmembrane currents were recorded at a holding potential of +50mV and −50 mV (with the cis side grounded) using a Tecella Triton 16channel amplifier at a low-pass filtering frequency of 3 kHz and asampling frequency of 10 kHz. Current traces were analyzed using theClampfit of the pClamp suite (Molecular Devices, USA).

Example 23

This example describes the structural and mechanistic insights into thebacterial amyloid secretion channel CsgG.Curli are functional amyloid fibres that constitute the major proteincomponent of the extracellular matrix in pellicle biofilms formed byBacteroidetes and Proteobacteria (predominantly of the a and cclasses)¹⁻³. They provide a fitness advantage in pathogenic strains andinduce a strong pro-inflammatory response during bacteraemia^(1,4,5).Curli formation requires a dedicated protein secretion machinerycomprising the outer membrane lipoprotein CsgG and two soluble accessoryproteins, CsgE and CsgF^(6,7). Here we report the X-ray structure ofEscherichia coli CsgGin a non-lipidated, soluble form as well as in itsNative membrane-extracted conformation. CsgG forms an oligomerictransport complex composed of nine anticodon-binding-domain-like unitsthat give rise to a 36-stranded β-barrel that traverses the bilayer andis connected to a cage-like vestibule in the periplasm. Thetransmembrane and periplasmic domains are separated by a 0.9-nm channelconstriction composed of three stacked concentric phenylalanine,asparagine and tyrosine rings that may guide the extended polypeptidesubstrate through the secretion pore. The specificity factor CsgE formsa nonameric adaptor that binds and closes off the periplasmic face ofthe secretion channel, creating a 24,000 Å³ pre-constriction chamber.Our structural, functional and electrophysiological analyses imply thatCsgG is an ungated, non-selective protein secretion channel that isexpected to employ a diffusion-based, entropy-driven transportmechanism.Curli are bacterial surface appendages that have structural and physicalcharacteristics of amyloid fibrils, best known from human degenerativediseases⁷⁻⁹. However, the role of bacterial amyloids such as curli areto facilitate biofilmformation^(4,10). Unlike pathogenic amyloids, whichare the product of protein misfolding, curli formation is coordinated byproteins encoded in two dedicated operons, csgBAC (curli specific genesBAC) and csgDEFG in Escherichia coli (FIG. 46)^(6,7). After secretion,CsgB nucleates CsgA subunits into curli flbres^(7,11,12). Secretion andextracellular deposition of CsgA and CsgB are dependent on two solubleaccessory factors, respectively CsgE and CsgF, as well as CsgG, a262-residue lipoprotein located in the outer membrane¹³⁻¹⁶. Because ofthe lack of hydrolysable energy sources or ion gradients at the outermembrane, CsgG falls into a specialized class of protein translocatorsthat must operate through an alternatively energized transportmechanism. In the absence of a structural model, the dynamic workings ofhow CsgG promotes the secretion and assembly of highly stableamyloidlike fibres in a regulated fashion across a biological membranehas so far remained enigmatic.Before insertion into the outer membrane, lipoproteins are pilotedacross the periplasm by means of the lipoprotein localization (Lol)pathway¹⁷. We observed that non-lipidated CsgG (CsgG_(C1S)) could beisolated as a soluble periplasmic intermediate, analogous to thepre-pore forms observed in pore-forming proteins and toxins¹⁸.CsgG_(C1S) was found predominantly as monomers, in addition to a minorfraction of discrete oligomeric complexes (FIG. 47)¹⁹. The solubleCsgG_(C1S) oligomers were crystallized and their structure wasdetermined to 2.8 Å, revealing a hexadecameric particle with eight-folddihedral symmetry (D8), consisting of two ring-shaped octamericcomplexes (C8) that are joined in a tail-to-tall interaction (FIG. 47and FIG. 46).The CsgG_(C1S) protomer shows an anticodon-binding domain(ABD)-like fold that is extended with two α-helices at the amino andcarboxy termini (αN and αC, respectively; FIG. 42 and FIG. 48a-c ).Additional CsgG-specific elements are an extended loop linking β1 andα1, two insertions in the loops connecting β3-β4 and β5-α3 and anextended α2 helix that is implicated in CsgG oligomerization by packingbetween adjacent monomers (FIG. 42b ). Further inter-protomer contactsare formed between the back of the β3-β5 sheet and the extended β1-α1loop (FIG. 48d, e ).In the CsgG_(C1S) structure, residues 1-17, which would link α1 to theN-terminal lipid anchor, are disordered and no obvious transmembrane(TM) domain can be discerned (FIG. 42). Attenuated total reflectionFourier transform infrared spectroscopy (ATR-FTIR) of CsgG_(C1S) andnative, membrane-extracted CsgG revealed that the latter has a higherabsorption in the β-sheet region (1,625-1,630 cm⁻¹) and a concomitantreduction in the random coil and α-helical regions (1,645-1,650 cm⁻¹ and1,656 cm⁻¹, respectively; FIG. 43a ), suggesting thatmembrane-associated CsgG contains a β-barrel domain. Candidate sequencestretches for β-strand formation are found in the poorly ordered,extended loops connecting β3-β4 (residues 134-154) and β5-α3 (residues184-204); deletion of these resulted in the loss of curli formation(FIG. 43b ).The crystal structure of detergent-extracted CsgG confirmeda conformational rearrangement of both regions into two adjacentβ-hairpins, extending the β-sheet formed by β3-β4 (TM1) and β5-α3 (TM2)(FIG. 43c ). Their juxtaposition in the CsgG oligomer gave rise to acomposite 36-stranded β-barrel (FIG. 43d ). Whereas the crystallizedCsgG_(C1S) oligomers showed a D8 symmetry, the CsgG structure showed D9symmetry, with CsgG protomers retaining equivalent interprotomercontacts, except for a 5° rotation relative to the central axis and a 4Å translation along the radial axes FIG. 47). This observation isreconciled in the in-solution oligomeric states revealed bysingle-particle electron microscopy, which exclusively found C9 and D9symmetries for membrane-extracted CsgG (FIG. 47). The predominantpresence of monomers in the non-lipidated sample and the symmetrymismatch with the membrane-bound protein argue that before membraneinsertion, CsgG is targeted to the outer membrane in a monomeric,LolA-bound form and that the C8 and D8 particles are an artefact ofhighly concentrated solutions of CsgG_(C1S). Furthermore, we show thatthe C9 nonamer rather than the D9 complex forms the physiologicallyrelevant particle, because in isolated E. coli outer membranes, cysteinesubstitutions in residues enclosed by the observed tail-to-taildimerization are accessible to labeling with maleimidepolyethyleneglycol (PEG, 5 kDa; FIG. 49).Thus, CsgG forms a nonameric transport complex 120 Å in width and 85 Åin height. The complex traverses the outer membrane through a36-stranded β-barrel with an inner diameter of 40 Å (FIG. 43e ). TheN-terminal lipid anchor is separated from the core domain by an18-residue linker that wraps over the adjacent protomer (FIG. 48d ). Thediacylglycerol- and amide-linked acyl chain on the N-terminal Cys arenot resolved in the electron density maps, but on the basis of thelocation of Leu 2 the lipid anchor is expected to flank the outer wallof the β-barrel. On the periplasmic side, the transporter forms a largesolvent-accessible cavity with an inner diameter of 35 Å and a height of40 Å that opens to the periplasmin a 50 Å mouth formed by helix 2 (FIG.43e ). At its apex, this periplasmic vestibule is separated from the TMchannel by a conserved 12-residue loop connecting β1 to α1 (C-loop;FIGS. 43e and 44 a, b), which constricts the secretion conduit to asolvent-excluded diameter of 9.0 Å (FIG. 44a, c ). These pore dimensionswould be compatible with the residence of one or two (for example alooped structure) extended polypeptide segments, with five residuesspanning the height of the constriction (FIG. 50). The luminal lining ofthe constriction is composed of three stacked concentric rings formed bythe side chains of residues Tyr 51, Asn 55 and Phe 56 (FIG. 44a, b ). Inthe anthrax PA63 toxin, a topologically equivalent concentric Phe ring(referred to as a ϕ-clamp) lines the entry of the translocation channeland catalyses polypeptide capture and passage²⁰⁻²². Multiple sequencealignment of CsgG-like translocators shows the absolute conservation ofPhe 56 and the conservative variation of Asn 55 to Ser or Thr (FIG. 51).Mutation of Phe 56 or Asn 55 to Ala leads to a near loss of curlproduction (FIG. 44d ), whereas a Asn 55→Ser substitution retainswild-type secretion levels, together alluding to the requirement of thestacked configuration of a ϕ-clamp followed by a hydrogen-bonddonor/acceptor in the CsgG constriction (FIG. 44b and FIG. 51).Single-channel current recordings of CsgG reconstituted in planarphospholipid bilayers led to a steady current of 43.1±4.5 pA (n=33) or−45.1±4.0 pA (n=13) using standard electrolyte conditions and apotential of +50 mV or −50 mV, respectively (FIG. 44e, f and FIG. 52).The observed current was in good agreement with the predicted value of46.6 pA calculated on the basis of a simple three-segment pore model andthe dimensions of the central constriction seen in the X-ray structure(FIG. 44c ). A second, low-conductance conformation can also be observedunder negative electrical field potential (−26.2±3.6 pA (n=13); FIG.52). It is unclear, however, whether this species is present underphysiological conditions.Our structural data and single-channel recordings imply that CsgG formsan ungated peptide diffusion channel. In PA₆₃, a model peptide diffusionchannel, polypeptide passage depends on a ΔpH-driven Brownian ratchetthat rectifies the diffusive steps in the translocation channel²⁰⁻²².However, such proton gradients are not present at the outer membrane,requiring an alternative driving force. Whereas at elevatedconcentrations CsgG facilitates a non-selective diffusive leakage ofperiplasmic polypeptides, secretion is specific for CsgA under nativeconditions and requires the periplasmic factor CsgE^(16,23). In thepresence of excess CsgE, purified CsgG forms a more slowly migratingspecies on native PAGE (FIG. 45a ). SDS-PAGE analysis shows this newspecies consists of a CsgG-CsgE complex that is present in a 1:1stoichiometry (FIG. 45b ). Cryoelectron microscopy (cryo-EM)visualization of CsgG-CsgE isolated by pull-down affinity purificationrevealed a nine-fold symmetrical particle corresponding to the CsgGnonamer and an additional capping density at the entrance to theperiplasmic vestibule, similar in size and shape to a C9 CsgE oligomeralso observed by single-particle EM and size-exclusion chromatography(FIG. 45c-e and FIG. 53). The location of the observed CsgG-CsgE contactinterface was corroborated by blocking point mutations in CsgG helix 2(FIG. 53). In agreement with a capping function, single-channelrecordings showed that CsgE binding to the translocator led to thespecific silencing of its ion conductance (FIG. 45f and FIG. 52). ThisCsgE capping of the channel seemed to be an all-or-none response infunction of CsgE nomamerbinding. At saturation, CsgE binding inducedfull blockage of the channel, whereas at about 10 nM, an equilibriumbetween CsgE binding and dissociation events resulted in anintermittently blocked or fully open translocator. At 1 nM or below,transient (<1 ms) partial blockage events may have stemmed fromshort-lived encounters with monomeric CsgE.Thus, CsgG and CsgE seem to form an encaging complex enclosing a centralcavity of ˜24,000 Å³, reminiscent in appearance to the substrate bindingcavity and encapsulating lid structure seen in the GroEL chaperonin andGroES co-chaperonin²⁴. The CsgG-CsgE enclosure would be compatible withthe full or partial entrapment of the 129-residue CsgA. The caging of atranslocation substrate has recently been observed in ABC toxins²⁵.Spatial confinement of an unfolded polypeptide leads to a decrease inits conformational space, creating an entropic potential that has beenshown to favour polypeptide folding in the case of chaperonins^(24,26).Similarly, we speculate that in curli transport the local highconcentration and conformational confinement of curli subunits in theCsgG vestibule would generate an entropic free-energy gradient over thetranslocation channel (FIG. 45g ). On capture into the constriction, thepolypeptide chain is then expected to move progressively outwards byBrownian diffusion, rectified by the entropic potential generated fromthe CsgE-mediated confinement and/or substrate trapping near thesecretion channel. For full confinement in the pre-constriction cavity,the escape of an unfolded 129-residue polypeptide to the bulk solventwould correspond to an entropic free-energy release of up to ˜80 kcalmol⁻¹ (about 340 kJ mol⁻¹; ref. 27). The initial entropic cost ofsubstrate docking and confinement are likely to be at least partlycompensated for by binding energy released during assembly of theCsgG-CsgE-CsgA complex and an already lowered CsgA entropy in theperiplasm. On theoretical grounds, three potential routes of CsgArecruitment to the secretion complex can be envisaged (FIG. 54).Curli-induced biofilms form a fitness and virulence factor in pathogenicEnterobacteriaceae^(4,5). Their unique secretion and assembly propertiesare also rapidly gaining interest for (bio)technologicalapplication^(23,28,29). Our structural characterization and biochemicalstudy of two key secretion components provide a tentative model of aniterative mechanism for the membrane translocation of unfolded proteinsubstrates in the absence of a hydrolysable energy source, a membranepotential or an ion gradient (FIG. 45e and FIG. 54). The full validationand deconstruction of the contributing factors in the proposed secretionmodel will require the in vitro reconstitution of the translocator toallow transport kinetics to be followed accurately at thesingle-molecule level.

Methods Cloning and Strains.

Expression constructs for the production of outer membrane localizedC-terminally StrepII-tagged CsgG (pPG1) and periplasmic C-terminallyStrepII-tagged CsgG_(C1S) (pPG2) have been described in ref. 19. Forselenomethionine labelling, StrepII-tagged CsgG_(C1S) was expressed inthe cytoplasm because of increased yields. Therefore, _(p)PG2 wasaltered to remove the N-terminal signal peptide using inverse PCR withprimers 5′-TCTTTAACCGCCCCGCCTAAAG-3′ (forward) and5′-CATTTTTTGCCCTCGTTATC-3′ (reverse) (pPG3). For phenotypic assays, acsgG deletion mutant of E. coli BW25141 (E. coli NVG2) was constructedby the method described in ref. 30 (with primers 5′-AATAACTCAACC GAT TTTTAA GCC CCA GCT TCA TAA GGA AAA TAA TCG TGT AGG CTG GAG CTG CTT C-3′ and5′-CGC TTA AAC AGT AAA ATG CCG GAT GAT AAT TCC GGC TTT TTT ATC TGC ATATGA ATA TCC TCC TTA G-3′). The various CsgG substitution mutants usedfor Cys accessibility assays and for phenotypic probing of the channelconstriction were constructed by site-directed mutagenesis (QuikChangeprotocol; Stratagene) starting from pMC2, a pTRC99a vector containingcsgG under control of the trc promoter¹⁴.

Protein Expression and Purification.

CsgG and CsgG_(C1S) were expressed and purified as described¹⁹. Inbrief, CsgG was recombinantly produced in E. coli BL 21 (DE3)transformed with pPG1 and extracted from isolated outer membranes withthe use of 1% n-dodecyl-b-D-maltoside (DDM) in buffer A (50 mM Tris-HClpH 8.0, 500 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT)).StrepII-tagged CsgG was loaded onto a 5 ml Strep-Tactin Sepharose column(Iba GmbH) and detergent exchanged by washing with 20 column volumes ofbuffer A supplemented with 0.5% tetraethylene glycol monooctyl ether(C8E4; Affymetrix) and 4 mM lauryldimethylamine-N-oxide (LDAO;Affymetrix). The protein was eluted by the addition of 2.5 mMD-desthiobiotin and concentrated to 5 mg ml⁻¹ for crystallizationexperiments. For selenomethionine labelling, CsgG_(C1S) was produced inthe Met auxotrophic strain B834 (DE3) transformed with pPG3 and grown onM9 minimal medium supplemented with 40 mg l⁻¹ L-selenomethionine. Cellpellets were resuspended in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mMEDTA, 5 mM DTT, supplemented with cOmplete Protease Inhibitor Cocktail(Roche) and disrupted by passage through a TS series cell disrupter(Constant Systems Ltd) operated at 20×10³ lb in⁻². Labelled CsgG_(C1S)was purified as described¹⁹. DTT (5 mM) was added throughout thepurification procedure to avoid oxidation of selenomethionine.

CsgE was produced in E. coli NEBC2566 cells harbouring pNH27 (ref. 16).Cell lysates in 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 25 mM imidazole, 5%(v/v) glycerol were loaded on a HisTrap FF (GE Healthcare). CsgE-his waseluted with a linear gradient to 500 mM imidazole in 20 mM Tris-HCl pH8.0, 150 mM NaCl, 5% (v/v) glycerol buffer. Fractions containing CsgEwere supplemented with 250 mM (NH₄)₂SO₄ and applied to a 5 ml HiTrapPhenyl HP column (GE Healthcare) equilibrated with 20 mM Tris-HCl pH8.0, 100 mM NaCl, 250 mM (NH₄)₂SO₄, 5% (v/v) glycerol. A linear gradientto 20 mM Tris-HCl pH 8.0, 10 mM NaCl, 5% (v/v) glycerol was applied forelution. CsgE containing fractions were loaded onto a Superose 6 PrepGrade 10/600 (GE Healthcare) column equilibrated in 20 mM Tris-HCl pH8.0, 100 mM NaCl, 5% (v/v) glycerol.

In-Solution Oligomeric State Assessment.

About 0.5 mg each of detergent-solubilized CsgG (0.5% C8E4, 4 mM LDAO)and CsgG_(C1S) were applied to a Superdex 200 10/300 GL analytical gelfiltration column (GE Healthcare) equilibrated with 25 mM Tris-HCl pH8.0, 500 mM NaCl, 1 mM DTT, 4 mM LDAO and 0.5% C8E4 (CsgG) or with 25 mMTris-HCl pH 8.0, 200 mM NaCl (CsgG_(C1S)), and run at 0.7 ml min⁻¹. Thecolumn elution volumes were calibrated with bovine thyroglobulin, bovineγ-globulin, chicken ovalbumin, horse myoglobulin and vitamin B12(Bio-Rad) (FIG. 47). Membrane-extracted CsgG, 20 mg of thedetergent-solubilized protein was also run on 3-10% blue native PAGEusing the procedure described in ref. 31 (FIG. 47). NativeMark (LifeTechnologies) unstained protein standard (7 ml) was used for molecularmass estimation.

Crystallization, Data Collection and Structure Determination.

Selenomethionine-labelled CsgG_(C1S) was concentrated to 3.8 mg ml⁻¹ andcrystallized by sitting-drop vapour diffusion against a solutioncontaining 100 mM sodium acetate pH 4.2, 8% PEG 4000 and 100 mM sodiummalonate pH 7.0. Crystals were incubated in crystallization buffersupplemented with 15% glycerol and flash-frozen in liquid nitrogen.Detergent-solubilized CsgG was concentrated to 5 mg ml⁻¹ andcrystallized by hanging-drop vapour diffusion against a solutioncontaining 100 mM Tris-HCl pH 8.0, 8% PEG 4000, 100 mM NaCl and 500 mMMgCl₂. Crystals were flash-frozen in liquid nitrogen and cryoprotectedby the detergent present in the crystallization solution. Foroptimization of crystal conditions and screening for crystals with gooddiffraction quality, crystals were analysed on beamlines Proxima-1 andProxima-2a (Soleil, France), PX-I (Swiss Light Source, Switzerland),I02, I03, I04 and I24 (Diamond Light Source, UK) and ID14eh2, ID23eh1and ID23eh2 (ESRF, France). Final diffraction data used for structuredetermination of CsgG_(C1S) and CsgG were collected at beamlines I04 andI03, respectively (see FIG. 55a for data collection and refinementstatistics). Diffraction data for CsgG_(C1S) were processed using Xia2and the XDS package.^(32,33). Crystals of CsgG_(C1S) belonged to spacegroup P1 with unit cell dimensions of a=101.3 Å, b=103.6 Å, c=141.7 Å,α=111.3°, β=90.5°, γ=118.2°, containing 16 protein copies in theasymmetric unit. For structure determination and refinement, datacollected at 0.9795 Å wavelength were truncated at 2.8 Å on the basis ofan I/δI cutoff of 2 in the highest-resolution shell. The structure wassolved using experimental phases calculated from a single anomalousdispersion (SAD) experiment. A total of 92 selenium sites were locatedin the asymmetric unit by using ShelxC and ShelxD³⁴, and were refinedand used for phase calculation with Sharp³⁵ (phasing power 0.79, figureof merit (FOM) 0.25). Experimental phases were density modified andaveraged by non-crystallographic symmetry (NCS) using Parrot³⁶ (FIG. 55;FOM 0.85). An initial model was built with Buccaneer³⁷ and refined byiterative rounds of maximum-likelihood refinement with Phenix refine³⁸and manual inspection and model (re)building in Coot³⁹. The finalstructure contained 28,853 atoms in 3,700 residues belonging to 16CsgG_(C1S) chains (FIG. 47), with a molprobity⁴⁰ score of 1.34; 98% ofthe residues lay in favoured regions of the Ramachandran plot (99.7% inallowed regions). Electron density maps showed no unambiguous densitycorresponding to possible solvent molecules, and no water molecules orions were therefore built in. Sixteenfold NCS averaging was maintainedthroughout refinement, using strict and local NCS restraints in earlyand late stages of refinement, respectively.

Diffraction data for CsgG were collected from a single crystal at 0.9763Å wavelength and were indexed and scaled, using the XDS package^(32,33),in space group C2 with unit-cell dimensions a=161.7 Å, b=372.3 Å,c=161.8 Å and β=92.9°, encompassing 18 CsgG copies in the asymmetricunit and a 72% solvent content. Diffraction data for structuredetermination and refinement were elliptically truncated to resolutionlimits of 3.6 Å, 3.7 Å and 3.8 Å along reciprocal cell directions a*, b*and c* and scaled anisotropically with the Diffraction AnisotropyServer⁴¹. Molecular replacement using the CsgG_(C1S) monomer provedunsuccessful. Analysis of the self rotation function revealed D₉symmetry in the asymmetric unit (not shown). On the basis of on theCsgG_(C1S) structure, a nonameric search model was generated in theassumption that after going from a C₈ to C₉ oligomer, the interprotomerarc at the particle circumference would stay approximately the same asthe interprotomer angle changed from 45° to 40°, giving a calculatedincrease in radius of about 4 Å. Using the calculated nonamer as searchmodel, a molecular replacement solution containing two copies was foundwith Phaser⁴². Inspection of density-modified and NCS-averaged electrondensity maps (Parrot³⁶; FIG. 55) allowed manual building of the TM1 andTM2 and remodeling of adjacent residues in the protein core, as well asthe building of residues 2-18, which were missing from the CsgG_(C1S)model and linked the α1 helix to the N-terminal lipid anchor. Refinementof the CsgG mode I was performed with Buster-TNT⁴³ and Refmac5 (ref. 44)for initial and final refinement rounds, respectively. Eighteenfoldlocal NCS restraints were applied throughout refinement, and Refmac5 wasrun employing a jelly-body refinement with sigma 0.01 and hydrogen-bondrestraints generated by Prosmart⁴⁵. The final structure contained 34,165atoms in 4,451 residues belonging to 18 CsgG chains (FIG. 47), with amolprobity score of 2.79; 93.0% of the residues lay in favoured regionsof the Ramachandran plot (99.3% in allowed regions). No unambiguouselectron density corresponding the N-terminal lipid anchor could bediscerned.

Congo Red Assay.

For analysis of Congo red binding, a bacterial overnight culture grownat 37° C. in Lysogeny Broth (LB) was diluted in LB medium until a D₆₀₀of 0.5 was reached. A 5 μl sample was spotted on LB agar platessupplemented with ampicillin (100 mg l⁻¹), Congo red (100 mg l⁻¹) and0.1% (w/v) isopropyl β-D-thiogalactoside (IPTG). Plates were incubatedat room temperature (20-22° C.) for 48 h to induce curl expression. Thedevelopment of the colony morphology and dye binding were observed at 48h.

Cysteine Accessibility Assays.

Cysteine mutants were generated in pMC2 using site-directed mutagenesisand expressed in E. coli LSR12 (ref. 7). Bacterial cultures grownovernight were spotted onto LB agar plates containing 1 mM IPTG and 100mg l⁻¹ ampicillin. Plates were incubated at room temperature and cellswere scraped after 48 h, resuspended in 1 ml of PBS and normalized usingD₆₀₀. The cells were lysed by sonication and centrifuged for 20 s at3,000 g at 4° C. to remove unbroken cells from cell lysate and suspendedmembranes. Proteins in the supernatant were labelled with 15 mMmethoxypolyethylene glycol-maleimide (MAL-PEG 5 kDa) for 1 h at roomtemperature. The reaction was stopped with 100 mM DTT and centrifuged at40,000 r.p.m. (˜100,000 g) in a 50.4 Ti rotor for 20 min at 4° C. topellet total membranes. The pellet was washed with 1% sodiumlauroylsarcosinate to solubilize cytoplasmic membranes and centrifuged again.The resulting outer membranes were resuspended and solubilized using PBScontaining 1% DDM. Metal affinity pulldowns with nickel beads were usedfor SDS-PAGE and anti-His western blots. E. coli LSR12 cells with emptypMC2 vector were used as negative control.

ATR-FTIR Spectroscopy.

ATR-FTIR measurements were performed on an Equinox 55 infraredspectrophotometer (Bruker), continuously purged with dried air, equippedwith a liquid-nitrogen-refrigerated mercury cadmium telluride detectorand a Golden Gate reflectance accessory (Specac). The internalreflection element was a diamond crystal (2 mm×2 mm) and the beamincidence angle was 45°. Each purified protein sample (1 μl) was spreadat the surface of the crystal and dried under a gaseous nitrogen flow toform a film. Each spectrum, recorded at 2 cm⁻¹ resolution, was anaverage of 128 accumulations for improved signal-to-noise ratio. All thespectra were treated with water vapour contribution subtraction,smoothed at a final resolution of 4 cm⁻¹ by apodization and normalizedon the area of the Amide I band (1,700-1,600 cm⁻¹) to allow theircomparison⁴⁶.

Negative Stain EM and Symmetry Determination.

Negative stain EM was used to monitor in-solution oligomerization statesof CsgG, CsgG_(C1S) and CsgE. CsgE, CsgG_(C1S) and amphipol-bound CsgGwere adjusted to a concentration of 0.05 mg ml⁻¹ and applied toglow-discharged carbon-coated copper grids (CF-400; Electron MicroscopySciences). After 1 min incubation, samples were blotted, then washed andstained in 2% uranyl acetate. Images were collected on a Tecnai T12BioTWIN LaB6 microscope operating at a voltage of 120 kV, at amagnification of ×49,000 and defocus between 800 and 2,000 nm. Contrasttransfer function (CTF), phase flipping and particle selection wereperformed as described for cryo-EM. For membrane extracted CsgG,octadecameric particles (1,780 in all) were analysed separately fromnonamers and top views. For purified CsgE a total of 2,452 particleswere analysed. Three-dimensional models were obtained as described forthe CsgG-CsgE cryo-EManalysis below and refined by several rounds ofmulti-reference alignment (MRA), multi-statistical analysis (MSA) andanchor set refinement in all cases, after normalization and centring,images were classified using IMAGIC-4D as described in the cryo-EMsection. The best classes corresponding to characteristic views wereselected for each set of particles. Symmetry determination of CsgG topviews was performed using the best class averages with roughly 20 imagesper class. The rotational autocorrelation function was calculated usingIMAGIC and plotted.

CsgG-CsgE Complex Formation.

For CsgG-CsgE complex formation, the solubilizing detergents in purifiedCsgG were exchanged for Amphipols A8-35 (Anatrace) by adding 120 μl ofCsgG (24 mg ml⁻¹ protein in 0.5% C8E4, 4 mM LDAO, 25 mM Tris-HCl pH 8.0,500 mM NaCl, 1 mM DTT) to 300 μl of detergent-destabilized liposomes (1mg ml⁻¹ 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 0.4%LDAO) and incubating for 5 min on ice before the addition of 90 ml ofA8-35 amphipols at 100 mg ml⁻¹ stock. After an additional 15 minincubation on ice, the sample was loaded on a Superose 6 10/300 GL (GEHealthcare) column and gel filtration was performed in 200 mM NaCl, 2.5%xylitol, 25 mM Tris-HCl pH 8, 0.2 mM DTT. An equal volume of purifiedmonomeric CsgE in 200 mM NaCl, 2.5% xylitol, 25 mM Tris-HCl pH 8, 0.2 mMDTT was added to the amphipol-solubilized CsgG at final proteinconcentrations of 15 and 5 μM for CsgE and CsgG, respectively, and thesample was run at 125V at 18° C. on a 4.5% native PAGE in 0.5×TBEbuffer. For the second, denaturing dimension, the band corresponding tothe CsgG-CsgE complex was cut out of unstained lanes run in parallel onthe same gel, boiled for 5 min in Laemmli buffer (60 mM Tris-HCl pH 6.8,2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.01% bromophenol blue) andrun on 4-20% SDS-PAGE. Purified CsgE and CsgG were run alongside thecomplex as control samples. Gels were stained with InstantBlue Coomassiefor visual inspection or SYPRO orange for stoichiometry assessment ofthe CsgG-CsgE complex by fluorescence detection (Typhoon FLA 9000) ofthe CsgE and CsgG bands on SDS-PAGE, yielding a CsgG/CsgE ratio of 0.97.

CsgG-CsgE Cryo-EM.

Cryo-electron microscopy was used to determine the in-solution structureof the C₉ CsgG-CsgE complex. CsgG-CsgE complex prepared as describedabove was bound and eluted from a HisTrap FF (GE Healthcare) to removeunbound CsgG, and on elution it was immediately applied to QuantifoilR2/2 carbon coated grids (Quantifoil Micro Tools GmbH) that had beenglow-discharged at 20 mA for 30 s. Samples were plunge-frozen in liquidnitrogen using an automated system (Leica) and observed under a FEI F20microscope operating at a voltage of 200 kV, a nominal magnification of×50,000 under low-dose conditions and a defocus range of 1.4-3 μm. Imageframes were recorded on a Falcon II detector. The pixel size at thespecimen level was 1.9 Å per pixel. The CTF parameters were assessedusing CTFFIND3 (ref. 47), and the phase flipping was done in SPIDER⁴⁶.Particles were automatically selected from CTF-corrected micrographsusing BOXER (EMAN2; ref. 49). Images with an astigmatism of more than10% were discarded. A total of 1,221 particles were selected from 75micrographs and windowed into 128-pixel×128-pixel boxes. Images werenormalized to the same mean and standard deviation and high-passfiltered at a low-resolution cut-off of ˜200 Å. They were centred andthen subjected to a first round of MSA. An initial reference set wasobtained using reference free classification in IMAGIC-4D (Image ScienceSoftware). The best classes corresponding to characteristic side viewsof the C₉ cylindrical particles were used as references for the MRA. ForCsgG-CsgE complex, the first three-dimensional model was calculated fromthe best 125 characteristic views (with good contrast and well-definedfeatures) encompassing 1,221 particles of the complex with orientationsdetermined by angular reconstitution (Image Science Software). Thethree-dimensional map was refined by iterative rounds of MRA, MSA andanchor set refinement. The resolution was estimated to be 24 Å byFourier shell correlation (FSC) according to the 0.5 criteria level FIG.52). Visualization of the map and figures was performed in UCSFChimera⁵⁰.

Bile Salt Toxicity Assay.

Outer-membrane permeability was investigated by decreased growth on agarplates containing bile salts. Tenfold serial dilutions of E. coli LSR12(ref. 7) cells (5 μl) harbouring both pLR42 (ref. 16) and pMC2 (ref. 14)(or derived helix 2 mutants) were spotted on McConkey agar platescontaining 100 μg l⁻¹ ampicillin, 25 μg l⁻¹ chloramphenicol, 1 mM IPTGwith or without 0.2% (w/v) L-arabinose. After incubation overnight at37° C., colony growth was examined.

Single-Channel Current Recordings.

Single-channel current recordings were performed using parallelhigh-resolution electrical recording with the Orbit 16 kit from Nanion.In brief, horizontal bilayers of1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids) wereformed over microcavities (of subpicolitre volume) in a 16-channelmultielectrode cavity array (MECA) chip (Ionera)⁵¹. Both the cis andtrans cavities above and below the bilayer contained 1.0 M KCl, 25 mMTris-HCl pH 8.0. To insert channels into the membrane, CsgG dissolved in25 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM DTT, 0.5% C8E4, 5 mM LDAO wasadded to the cis compartment to a final concentration of 90-300 nM. Totest the interaction of the CsgG channel with CsgE, a solution of thelatter protein dissolved in 25 mM Tris-HCl pH 8.0, 150 mM NaCl was addedto the cis compartment to final concentrations of 0.1, 1, 10 and 100 nM.Transmembrane currents were recorded at a holding potential of +50 mVand −50 mV (with the cis side grounded) using a Tecella Triton16-channel amplifier at a low-pass filtering frequency of 3 kHz and asampling frequency of 10 kHz. Current traces were analysed using theClampfit of the pClamp suite (Molecular Devices). Plots were generatedusing Origin 8.6 (Microcal)⁵².

Measured currents were compared with those calculated based on the poredimensions of the CsgGX-ray structure, modelled to be composed of threesegments: the transmembrane section, the periplasmic vestibule, and theinner channel constriction connecting the two. The first two segmentswere modeled to be of conical shape; the constriction was represented asa cylinder. The corresponding resistances R1, R2 and R3, respectively,were calculated as

R1=L1/(πD ₁ d ₁κ)

R2=L2/(πD ₂ d ₂κ)

R3=L3/(πD ₁ d ₂κ)

where L1, L2 and L3 are the axial lengths of the segments, measuring3.5, 4.0 and 2.0 nm, respectively, and D1, d1, D2 and d2 are the maximumand minimum diameters of segments 1 and 2, measuring 4.0, 0.8, 3.4 and0.8 nm, respectively. The conductivity κ has a value of 10.6 Sm⁻¹. Thecurrent was calculated by inserting R1, R2 and R3 and voltage V=50 mVinto

I=V/(R1+R2+R3)

Access resistance was not found to alter the predicted currentsignificantly.

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1-76. (canceled)
 77. An apparatus comprising a transmembrane proteinpore inserted into an in vitro membrane, wherein the transmembraneprotein pore comprises at least one CsgG monomer having amino acidmutations at two or more positions corresponding to Y51, N55, and F56 ofthe sequence shown in SEQ ID NO:
 390. 78. The apparatus of claim 77,wherein the at least one CsgG monomer comprises mutations at positionscorresponding to Y51/N55, Y51/F56, N55/F56 or Y51/N55/F56.
 79. Theapparatus of claim 77, wherein the at least one CsgG monomer comprisesmutations corresponding to F56N/N55Q, F56N/N55R, F56N/N55K, F56N/N55S,F56N/N55G, F56N/N55A, F56N/N55T, F56Q/N55Q, F56Q/N55R, F56Q/N55K,F56Q/N55S, F56Q/N55G, F56Q/N55A, F56Q/N55T, F56R/N55Q, F56R/N55R,F56R/N55K, F56R/N55S, F56R/N55G, F56R/N55A, F56R/N55T, F56S/N55Q,F56S/N55R, F56S/N55K, F56S/N55S, F56S/N55G, F56S/N55A, F56S/N55T,F56G/N55Q, F56G/N55R, F56G/N55K, F56G/N55S, F56G/N55G, F56G/N55A,F56G/N55T, F56A/N55Q, F56A/N55R, F56A/N55K, F56A/N55S, F56A/N55G,F56A/N55A, F56A/N55T, F56K/N55Q, F56K/N55R, F56K/N55K, F56K/N55S,F56K/N55G, F56K/N55A, F56K/N55T, F56N/Y51L, F56N/Y51V, F56N/Y51A,F56N/Y51N, F56N/Y51Q, F56N/Y51S, F56N/Y51G, F56Q/Y51L, F56Q/Y51V,F56Q/Y51A, F56Q/Y51N, F56Q/Y51Q, F56Q/Y51S, F56Q/Y51G, F56R/Y51L,F56R/Y5N, F56R/Y51Q, F56R/Y51A, F56R/Y5N, F56R/Y51Q, F56R/Y51S,F56R/Y51G, F56S/Y51L, F56S/Y51V, F56S/Y51A, F56S/Y51N, F56S/Y51Q,F56S/Y51S, F56S/Y51G, F56G/Y51L, F56G/Y51V, F56G/Y51A, F56G/Y51N,F56G/Y51Q, F56G/Y51S, F56G/Y51G, F56A/Y51L, F56A/Y51V, F56A/Y51A,F56A/Y51N, F56A/Y51Q, F56A/Y51S, F56A/Y51G, F56K/Y51L, F56K/Y51V,F56K/Y51A, F56K/Y51N, F56K/Y51Q, F56K/Y51S, F56K/Y51G, N55Q/Y51L,N55Q/Y51V, N55Q/Y51A, N55Q/Y51N, N55Q/Y51Q, N55Q/Y51S, N55Q/Y51G,N55R/Y51L, N55R/Y51V, N55R/Y51A, N55R/Y51N, N55R/Y51Q, N55R/Y51S,N55R/Y51G, N55K/Y51L, N55K/Y51V, N55K/Y51A, N55K/Y51N, N55K/Y51Q,N55K/Y51S, N55K/Y51G, N55S/Y51L, N55S/Y51V, N55S/Y51A, N55S/Y51N,N55S/Y51Q, N55S/Y51S, N55S/Y51G, N55G/Y51L, N55G/Y51V, N55G/Y51A,N55G/Y51N, N55G/Y51Q, N55G/Y51S, N55G/Y51G, N55A/Y51L, N55A/Y51V,N55A/Y51A, N55A/Y51N, N55A/Y51Q, N55A/Y51S, N55A/Y51G, N55T/Y51L,N55T/Y51V, N55T/Y51A, N55T/Y51N, N55T/Y51Q, N55T/Y51S, N55T/Y51G,F56N/N55Q/Y51L, F56N/N55Q/Y51V, F56N/N55Q/Y51A, F56N/N55Q/Y51N,F56N/N55Q/Y51Q, F56N/N55Q/Y51S, F56N/N55Q/Y51G, F56N/N55R/Y51L,F56N/N55R/Y51V, F56N/N55R/Y51A, F56N/N55R/Y51N, F56N/N55R/Y51Q,F56N/N55R/Y51S, F56N/N55R/Y51G, F56N/N55K/Y51L, F56N/N55K/Y51V,F56N/N55K/Y51A, F56N/N55K/Y51N, F56N/N55K/Y51Q, F56N/N55K/Y51S,F56N/N55K/Y51G, F56N/N55S/Y51L, F56N/N55S/Y51V, F56N/N55S/Y51A,F56N/N55S/Y51N, F56N/N55S/Y51Q, F56N/N55S/Y51S, F56N/N55S/Y51G,F56N/N55G/Y51L, F56N/N55G/Y51V, F56N/N55G/Y51A, F56N/N55G/Y51N,F56N/N55G/Y51Q, F56N/N55G/Y51S, F56N/N55G/Y51G, F56N/N55A/Y51L,F56N/N55A/Y51V, F56N/N55A/Y51A, F56N/N55A/Y51N, F56N/N55A/Y51Q,F56N/N55A/Y51S, F56N/N55A/Y51G, F56N/N55T/Y51L, F56N/N55T/Y51V,F56N/N55T/Y51A, F56N/N55T/Y51N, F56N/N55T/Y51Q, F56N/N55T/Y51S,F56N/N55T/Y51G, F56Q/N55Q/Y51L, F56Q/N55Q/Y51V, F56Q/N55Q/Y51A,F56Q/N55Q/Y51N, F56Q/N55Q/Y51Q, F56Q/N55Q/Y51S, F56Q/N55Q/Y51G,F56Q/N55R/Y51L, F56Q/N55R/Y51V, F56Q/N55R/Y51A, F56Q/N55R/Y51N,F56Q/N55R/Y51Q, F56Q/N55R/Y51S, F56Q/N55R/Y51G, F56Q/N55K/Y51L,F56Q/N55K/Y51V, F56Q/N55K/Y51A, F56Q/N55K/Y51N, F56Q/N55K/Y51Q,F56Q/N55K/Y51S, F56Q/N55K/Y51G, F56Q/N55S/Y51L, F56Q/N55S/Y51V,F56Q/N55S/Y51A, F56Q/N55S/Y51N, F56Q/N55S/Y51Q, F56Q/N55S/Y51S,F56Q/N55S/Y51G, F56Q/N55G/Y51L, F56Q/N55G/Y51V, F56Q/N55G/Y51A,F56Q/N55G/Y51N, F56Q/N55G/Y51Q, F56Q/N55G/Y51S, F56Q/N55G/Y51G,F56Q/N55A/Y51L, F56Q/N55A/Y51V, F56Q/N55A/Y51A, F56Q/N55A/Y51N,F56Q/N55A/Y51Q, F56Q/N55A/Y51S, F56Q/N55A/Y51G, F56Q/N55T/Y51L,F56Q/N55T/Y51V, F56Q/N55T/Y51A, F56Q/N55T/Y51N, F56Q/N55T/Y51Q,F56Q/N55T/Y51S, F56Q/N55T/Y51G, F56R/N55Q/Y51L, F56R/N55Q/Y51V,F56R/N55Q/Y51A, F56R/N55Q/Y51N, F56R/N55Q/Y51Q, F56R/N55Q/Y51S,F56R/N55Q/Y51G, F56R/N55R/Y51L, F56R/N55R/Y51V, F56R/N55R/Y51A,F56R/N55R/Y51N, F56R/N55R/Y51Q, F56R/N55R/Y51S, F56R/N55R/Y51G,F56R/N55K/Y51L, F56R/N55K/Y51V, F56R/N55K/Y51A, F56R/N55K/Y51N,F56R/N55K/Y51Q, F56R/N55K/Y51S, F56R/N55K/Y51G, F56R/N55S/Y51L,F56R/N55S/Y51V, F56R/N55S/Y51A, F56R/N55S/Y51N, F56R/N55S/Y51Q,F56R/N55S/Y51S, F56R/N55S/Y51G, F56R/N55G/Y51L, F56R/N55G/Y51V,F56R/N55G/Y51A, F56R/N55G/Y51N, F56R/N55G/Y51Q, F56R/N55G/Y51S,F56R/N55G/Y51G, F56R/N55A/Y51L, F56R/N55A/Y51V, F56R/N55A/Y51A,F56R/N55A/Y51N, F56R/N55A/Y51Q, F56R/N55A/Y51S, F56R/N55A/Y51G,F56R/N55T/Y51L, F56R/N55T/Y51V, F56R/N55T/Y51A, F56R/N55T/Y51N,F56R/N55T/Y51Q, F56R/N55T/Y51S, F56R/N55T/Y51G, F56S/N55Q/Y51L,F56S/N55Q/Y51V, F56S/N55Q/Y51A, F56S/N55Q/Y51N, F56S/N55Q/Y51Q,F56S/N55Q/Y51S, F56S/N55Q/Y51G, F56S/N55R/Y51L, F56S/N55R/Y51V,F56S/N55R/Y51A, F56S/N55R/Y51N, F56S/N55R/Y51Q, F56S/N55R/Y51S,F56S/N55R/Y51G, F56S/N55K/Y51L, F56S/N55K/Y51V, F56S/N55K/Y51A,F56S/N55K/Y51N, F56S/N55K/Y51Q, F56S/N55K/Y51S, F56S/N55K/Y51G,F56S/N55S/Y51L, F56S/N55S/Y51V, F56S/N55S/Y51A, F56S/N55S/Y51N,F56S/N55S/Y51Q, F56S/N55S/Y51S, F56S/N55S/Y51G, F56S/N55G/Y51L,F56S/N55G/Y51V, F56S/N55G/Y51A, F56S/N55G/Y51N, F56S/N55G/Y51Q,F56S/N55G/Y51S, F56S/N55G/Y51G, F56S/N55A/Y51L, F56S/N55A/Y51V,F56S/N55A/Y51A, F56S/N55A/Y51N, F56S/N55A/Y51Q, F56S/N55A/Y51S,F56S/N55A/Y51G, F56S/N55T/Y51L, F56S/N55T/Y51V, F56S/N55T/Y51A,F56S/N55T/Y51N, F56S/N55T/Y51Q, F56S/N55T/Y51S, F56S/N55T/Y51G,F56G/N55Q/Y51L, F56G/N55Q/Y51V, F56G/N55Q/Y51A, F56G/N55Q/Y51N,F56G/N55Q/Y51Q, F56G/N55Q/Y51S, F56G/N55Q/Y51G, F56G/N55R/Y51L,F56G/N55R/Y51V, F56G/N55R/Y51A, F56G/N55R/Y51N, F56G/N55R/Y51Q,F56G/N55R/Y51S, F56G/N55R/Y51G, F56G/N55K/Y51L, F56G/N55K/Y5V,F56G/N55K/Y51A, F56G/N55K/Y51N, F56G/N55K/Y51Q, F56G/N55K/Y51S,F56G/N55K/Y51G, F56G/N55S/Y51L, F56G/N55S/Y51V, F56G/N55S/Y51A,F56G/N55S/Y51N, F56G/N55S/Y51Q, F56G/N55S/Y51S, F56G/N55S/Y51G,F56G/N55G/Y51L, F56G/N55G/Y51V, F56G/N55G/Y51A, F56G/N55G/Y51N,F56G/N55G/Y51Q, F56G/N55G/Y51S, F56G/N55G/Y51G, F56G/N55A/Y51L,F56G/N55A/Y51V, F56G/N55A/Y51A, F56G/N55A/Y51N, F56G/N55A/Y51Q,F56G/N55A/Y51S, F56G/N55A/Y51G, F56G/N55T/Y51L, F56G/N55T/Y51V,F56G/N55T/Y51A, F56G/N55T/Y51N, F56G/N55T/Y51Q, F56G/N55T/Y51S,F56G/N55T/Y51G, F56A/N55Q/Y51L, F56A/N55Q/Y51V, F56A/N55Q/Y51A,F56A/N55Q/Y51N, F56A/N55Q/Y51Q, F56A/N55Q/Y51S, F56A/N55Q/Y51G,F56A/N55R/Y51L, F56A/N55R/Y51V, F56A/N55R/Y51A, F56A/N55R/Y51N,F56A/N55R/Y51Q, F56A/N55R/Y51S, F56A/N55R/Y51G, F56A/N55K/Y51L,F56A/N55K/Y51V, F56A/N55K/Y51A, F56A/N55K/Y51N, F56A/N55K/Y51Q,F56A/N55K/Y51S, F56A/N55K/Y51G, F56A/N55S/Y51L, F56A/N55S/Y51V,F56A/N55S/Y51A, F56A/N55S/Y51N, F56A/N55S/Y51Q, F56A/N55S/Y51S,F56A/N55S/Y51G, F56A/N55G/Y51L, F56A/N55G/Y51V, F56A/N55G/Y51A,F56A/N55G/Y51N, F56A/N55G/Y51Q, F56A/N55G/Y51S, F56A/N55G/Y51G,F56A/N55A/Y51L, F56A/N55A/Y51V, F56A/N55A/Y51A, F56A/N55A/Y51N,F56A/N55A/Y51Q, F56A/N55A/Y51S, F56A/N55A/Y51G, F56A/N55T/Y51L,F56A/N55T/Y51V, F56A/N55T/Y51A, F56A/N55T/Y51N, F56A/N55T/Y51Q,F56A/N55T/Y51S, F56A/N55T/Y51G, F56K/N55Q/Y51L, F56K/N55Q/Y51V,F56K/N55Q/Y51A, F56K/N55Q/Y51N, F56K/N55Q/Y51Q, F56K/N55Q/Y51S,F56K/N55Q/Y51G, F56K/N55R/Y51L, F56K/N55R/Y51V, F56K/N55R/Y51A,F56K/N55R/Y51N, F56K/N55R/Y51Q, F56K/N55R/Y51S, F56K/N55R/Y51G,F56K/N55K/Y51L, F56K/N55K/Y51V, F56K/N55K/Y51A, F56K/N55K/Y51N,F56K/N55K/Y51Q, F56K/N55K/Y51S, F56K/N55K/Y51G, F56K/N55S/Y51L,F56K/N55S/Y51V, F56K/N55S/Y51A, F56K/N55S/Y51N, F56K/N55S/Y51Q,F56K/N55S/Y51S, F56K/N55S/Y51G, F56K/N55G/Y51L, F56K/N55G/Y51V,F56K/N55G/Y51A, F56K/N55G/Y51N, F56K/N55G/Y51Q, F56K/N55G/Y51S,F56K/N55G/Y51G, F56K/N55A/Y51L, F56K/N55A/Y51V, F56K/N55A/Y51A,F56K/N55A/Y51N, F56K/N55A/Y51Q, F56K/N55A/Y51S, F56K/N55A/Y51G,F56K/N55T/Y51L, F56K/N55T/Y51V, F56K/N55T/Y51A, F56K/N55T/Y51N,F56K/N55T/Y51Q, F56K/N55T/Y51S, F56K/N55T/Y51G, F56E/N55R, F56E/N55K,F56D/N55R, F56D/N55K, F56R/N55E, F56R/N55D, F56K/N55E or F56K/N55D. 80.The apparatus of claim 77, wherein the transmembrane protein pore is ahetero-oligomeric pore.
 81. The apparatus of claim 80, wherein thehetero-oligomeric pore comprises nine CsgG monomers, and wherein atleast one of the nine CsgG monomers is different from the other CsgGmonomers.
 82. The apparatus of claim 80, wherein the hetero-oligomericpore comprises a monomer that is not a CsgG monomer.
 83. The apparatusof claim 77, wherein the transmembrane protein pore is a homo-oligomericpore.
 84. The apparatus of claim 83, wherein the homo-oligomeric poreconsists of nine identical CsgG monomers.
 85. A transmembrane proteinpore comprising at least one CsgG monomer, wherein the CsgG monomercomprises amino acid mutations at positions corresponding to Y51 and F56of the sequence shown in SEQ ID NO:
 390. 86. The pore of claim 85,further comprising a mutation at position corresponding to N55.
 87. Thepore of claim 85, wherein the at least one CsgG monomer comprisesmutations corresponding to F56N/Y51L, F56N/Y51V, F56N/Y51A, F56N/Y51N,F56N/Y51Q, F56N/Y51S, F56N/Y51G, F56Q/Y51L, F56Q/Y51V, F56Q/Y51A,F56Q/Y51N, F56Q/Y51Q, F56Q/Y51S, F56Q/Y51G, F56R/Y51L, F56R/Y51V,F56R/Y51A, F56R/Y51N, F56R/Y51Q, F56R/Y51S, F56R/Y51G, F56S/Y51L,F56S/Y51V, F56S/Y51A, F56S/Y51N, F56S/Y51Q, F56S/Y51S, F56S/Y51G,F56G/Y51L, F56G/Y51V, F56G/Y51A, F56G/Y51N, F56G/Y51Q, F56G/Y51S,F56G/Y51G, F56A/Y51L, F56A/Y51V, F56A/Y51A, F56A/Y51N, F56A/Y51Q,F56A/Y51S, F56A/Y51G, F56K/Y51L, F56K/Y51V, F56K/Y51A, F56K/Y51N,F56K/Y51Q, F56K/Y51S, F56K/Y51G, F56N/N55Q/Y51L, F56N/N55Q/Y51V,F56N/N55Q/Y51A, F56N/N55Q/Y51N, F56N/N55Q/Y51Q, F56N/N55Q/Y51S,F56N/N55Q/Y51G, F56N/N55R/Y51L, F56N/N55R/Y51V, F56N/N55R/Y51A,F56N/N55R/Y51N, F56N/N55R/Y51Q, F56N/N55R/Y51S, F56N/N55R/Y51G,F56N/N55K/Y51L, F56N/N55K/Y51V, F56N/N55K/Y51A, F56N/N55K/Y51N,F56N/N55K/Y51Q, F56N/N55K/Y51S, F56N/N55K/Y51G, F56N/N55S/Y51L,F56N/N55S/Y51V, F56N/N55S/Y51A, F56N/N55S/Y51N, F56N/N55S/Y51Q,F56N/N55S/Y51S, F56N/N55S/Y51G, F56N/N55G/Y51L, F56N/N55G/Y51V,F56N/N55G/Y51A, F56N/N55G/Y51N, F56N/N55G/Y51Q, F56N/N55G/Y51S,F56N/N55G/Y51G, F56N/N55A/Y51L, F56N/N55A/Y51V, F56N/N55A/Y51A,F56N/N55A/Y51N, F56N/N55A/Y51Q, F56N/N55A/Y51S, F56N/N55A/Y51G,F56N/N55T/Y51L, F56N/N55T/Y51V, F56N/N55T/Y51A, F56N/N55T/Y51N,F56N/N55T/Y51Q, F56N/N55T/Y51S, F56N/N55T/Y51G, F56Q/N55Q/Y51L,F56Q/N55Q/Y51V, F56Q/N55Q/Y51A, F56Q/N55Q/Y51N, F56Q/N55Q/Y51Q,F56Q/N55Q/Y51S, F56Q/N55Q/Y51G, F56Q/N55R/Y51L, F56Q/N55R/Y51V,F56Q/N55R/Y51A, F56Q/N55R/Y51N, F56Q/N55R/Y51Q, F56Q/N55R/Y51S,F56Q/N55R/Y51G, F56Q/N55K/Y51L, F56Q/N55K/Y51V, F56Q/N55K/Y51A,F56Q/N55K/Y51N, F56Q/N55K/Y51Q, F56Q/N55K/Y51S, F56Q/N55K/Y51G,F56Q/N55S/Y51L, F56Q/N55S/Y51V, F56Q/N55S/Y51A, F56Q/N55S/Y51N,F56Q/N55S/Y51Q, F56Q/N55S/Y51S, F56Q/N55S/Y51G, F56Q/N55G/Y51L,F56Q/N55G/Y51V, F56Q/N55G/Y51A, F56Q/N55G/Y51N, F56Q/N55G/Y51Q,F56Q/N55G/Y51S, F56Q/N55G/Y51G, F56Q/N55A/Y51L, F56Q/N55A/Y51V,F56Q/N55A/Y51A, F56Q/N55A/Y51N, F56Q/N55A/Y51Q, F56Q/N55A/Y51S,F56Q/N55A/Y51G, F56Q/N55T/Y51L, F56Q/N55T/Y51V, F56Q/N55T/Y51A,F56Q/N55T/Y51N, F56Q/N55T/Y51Q, F56Q/N55T/Y51S, F56Q/N55T/Y51G,F56R/N55Q/Y51L, F56R/N55Q/Y51V, F56R/N55Q/Y51A, F56R/N55Q/Y51N,F56R/N55Q/Y51Q, F56R/N55Q/Y51S, F56R/N55Q/Y51G, F56R/N55R/Y51L,F56R/N55R/Y51V, F56R/N55R/Y51A, F56R/N55R/Y51N, F56R/N55R/Y51Q,F56R/N55R/Y51S, F56R/N55R/Y51G, F56R/N55K/Y51L, F56R/N55K/Y51V,F56R/N55K/Y51A, F56R/N55K/Y51N, F56R/N55K/Y51Q, F56R/N55K/Y51S,F56R/N55K/Y51G, F56R/N55S/Y51L, F56R/N55S/Y51V, F56R/N55S/Y51A,F56R/N55S/Y51N, F56R/N55S/Y51Q, F56R/N55S/Y51S, F56R/N55S/Y51G,F56R/N55G/Y51L, F56R/N55G/Y51V, F56R/N55G/Y51A, F56R/N55G/Y51N,F56R/N55G/Y51Q, F56R/N55G/Y51S, F56R/N55G/Y51G, F56R/N55A/Y51L,F56R/N55A/Y51V, F56R/N55A/Y51A, F56R/N55A/Y51N, F56R/N55A/Y51Q,F56R/N55A/Y51S, F56R/N55A/Y51G, F56R/N55T/Y51L, F56R/N55T/Y51V,F56R/N55T/Y51A, F56R/N55T/Y51N, F56R/N55T/Y51Q, F56R/N55T/Y51S,F56R/N55T/Y51G, F56S/N55Q/Y51L, F56S/N55Q/Y51V, F56S/N55Q/Y51A,F56S/N55Q/Y51N, F56S/N55Q/Y51Q, F56S/N55Q/Y51S, F56S/N55Q/Y51G,F56S/N55R/Y51L, F56S/N55R/Y51V, F56S/N55R/Y51A, F56S/N55R/Y51N,F56S/N55R/Y51Q, F56S/N55R/Y51S, F56S/N55R/Y51G, F56S/N55K/Y51L,F56S/N55K/Y51V, F56S/N55K/Y51A, F56S/N55K/Y51N, F56S/N55K/Y51Q,F56S/N55K/Y51S, F56S/N55K/Y51G, F56S/N55S/Y51L, F56S/N55S/Y51V,F56S/N55S/Y51A, F56S/N55S/Y51N, F56S/N55S/Y51Q, F56S/N55S/Y51S,F56S/N55S/Y51G, F56S/N55G/Y51L, F56S/N55G/Y51V, F56S/N55G/Y51A,F56S/N55G/Y51N, F56S/N55G/Y51Q, F56S/N55G/Y51S, F56S/N55G/Y51G,F56S/N55A/Y51L, F56S/N55A/Y51V, F56S/N55A/Y51A, F56S/N55A/Y51N,F56S/N55A/Y51Q, F56S/N55A/Y51S, F56S/N55A/Y51G, F56S/N55T/Y51L,F56S/N55T/Y51V, F56S/N55T/Y51A, F56S/N55T/Y51N, F56S/N55T/Y51Q,F56S/N55T/Y51S, F56S/N55T/Y51G, F56G/N55Q/Y51L, F56G/N55Q/Y51V,F56G/N55Q/Y51A, F56G/N55Q/Y51N, F56G/N55Q/Y51Q, F56G/N55Q/Y51S,F56G/N55Q/Y51G, F56G/N55R/Y51L, F56G/N55R/Y51V, F56G/N55R/Y51A,F56G/N55R/Y51N, F56G/N55R/Y51Q, F56G/N55R/Y51S, F56G/N55R/Y51G,F56G/N55K/Y51L, F56G/N55K/Y51V, F56G/N55K/Y51A, F56G/N55K/Y51N,F56G/N55K/Y51Q, F56G/N55K/Y51S, F56G/N55K/Y51G, F56G/N55S/Y51L,F56G/N55S/Y51V, F56G/N55S/Y51A, F56G/N55S/Y51N, F56G/N55S/Y51Q,F56G/N55S/Y51S, F56G/N55S/Y51G, F56G/N55G/Y51L, F56G/N55G/Y51V,F56G/N55G/Y51A, F56G/N55G/Y51N, F56G/N55G/Y51Q, F56G/N55G/Y51S,F56G/N55G/Y51G, F56G/N55A/Y51L, F56G/N55A/Y51V, F56G/N55A/Y51A,F56G/N55A/Y51N, F56G/N55A/Y51Q, F56G/N55A/Y51S, F56G/N55A/Y51G,F56G/N55T/Y51L, F56G/N55T/Y51V, F56G/N55T/Y51A, F56G/N55T/Y51N,F56G/N55T/Y51Q, F56G/N55T/Y51S, F56G/N55T/Y51G, F56A/N55Q/Y51L,F56A/N55Q/Y51V, F56A/N55Q/Y51A, F56A/N55Q/Y51N, F56A/N55Q/Y51Q,F56A/N55Q/Y51S, F56A/N55Q/Y51G, F56A/N55R/Y51L, F56A/N55R/Y51V,F56A/N55R/Y51A, F56A/N55R/Y51N, F56A/N55R/Y51Q, F56A/N55R/Y51S,F56A/N55R/Y51G, F56A/N55K/Y51L, F56A/N55K/Y51V, F56A/N55K/Y51A,F56A/N55K/Y51N, F56A/N55K/Y51Q, F56A/N55K/Y51S, F56A/N55K/Y51G,F56A/N55S/Y51L, F56A/N55S/Y51V, F56A/N55S/Y51A, F56A/N55S/Y51N,F56A/N55S/Y51Q, F56A/N55S/Y51S, F56A/N55S/Y51G, F56A/N55G/Y51L,F56A/N55G/Y51V, F56A/N55G/Y51A, F56A/N55G/Y51N, F56A/N55G/Y51Q,F56A/N55G/Y51S, F56A/N55G/Y51G, F56A/N55A/Y51L, F56A/N55A/Y51V,F56A/N55A/Y51A, F56A/N55A/Y51N, F56A/N55A/Y51Q, F56A/N55A/Y51S,F56A/N55A/Y51G, F56A/N55T/Y51L, F56A/N55T/Y51V, F56A/N55T/Y51A,F56A/N55T/Y51N, F56A/N55T/Y51Q, F56A/N55T/Y51S, F56A/N55T/Y51G,F56K/N55Q/Y51L, F56K/N55Q/Y51V, F56K/N55Q/Y51A, F56K/N55Q/Y51N,F56K/N55Q/Y51Q, F56K/N55Q/Y51S, F56K/N55Q/Y51G, F56K/N55R/Y51L,F56K/N55R/Y51V, F56K/N55R/Y51A, F56K/N55R/Y51N, F56K/N55R/Y51Q,F56K/N55R/Y51S, F56K/N55R/Y51G, F56K/N55K/Y51L, F56K/N55K/Y51V,F56K/N55K/Y51A, F56K/N55K/Y51N, F56K/N55K/Y51Q, F56K/N55K/Y51S,F56K/N55K/Y51G, F56K/N55S/Y51L, F56K/N55S/Y51V, F56K/N55S/Y51A,F56K/N55S/Y51N, F56K/N55S/Y51Q, F56K/N55S/Y51S, F56K/N55S/Y51G,F56K/N55G/Y51L, F56K/N55G/Y51V, F56K/N55G/Y51A, F56K/N55G/Y51N,F56K/N55G/Y51Q, F56K/N55G/Y51S, F56K/N55G/Y51G, F56K/N55A/Y51L,F56K/N55A/Y51V, F56K/N55A/Y51A, F56K/N55A/Y51N, F56K/N55A/Y51Q,F56K/N55A/Y51S, F56K/N55A/Y51G, F56K/N55T/Y51L, F56K/N55T/Y51V,F56K/N55T/Y51A, F56K/N55T/Y51N, F56K/N55T/Y51Q, F56K/N55T/Y51S, orF56K/N55T/Y51G.
 88. The pore of claim 85, wherein the pore is ahetero-oligomeric pore.
 89. The pore of claim 88, wherein thehetero-oligomeric pore comprises nine CsgG monomers, and wherein atleast one of the nine CsgG monomers is different from the other CsgGmonomers.
 90. The pore of claim 88, wherein the hetero-oligomeric porecomprises a monomer that is not a CsgG monomer.
 91. The pore of claim85, wherein the pore is a homo-oligomeric pore.
 92. The pore of claim91, wherein the homo-oligomeric pore consists of nine identical CsgGmonomers.
 93. An apparatus produced by a method comprising: (i)obtaining a transmembrane protein pore comprising at least one CsgGmonomer, wherein the CsgG monomer comprises amino acid mutations at oneor more of positions Y51, N55, and F56 corresponding to the sequenceshown in SEQ ID NO: 390; and (ii) contacting the pore with an in vitromembrane such that the pore is inserted in the in vitro membrane.
 94. Amethod for identifying a transmembrane protein pore type, the methodcomprising: (i) applying a voltage current to a solution containing atransmembrane protein pore inserted into an artificial membrane; (ii)measuring an ion current flow through the transmembrane protein pore;(iii) adding a Curli formation accessory protein to the solution underconditions under which the accessory protein contacts the transmembraneprotein pore; and (iv) identifying the transmembrane protein pore as aCsgG pore if the ion current flow is modified compared to the ioncurrent flow in step (ii).
 95. The method of claim 94, wherein the ioncurrent flow in step (iv) is blocked.
 96. The method of claim 94,wherein the Curli formation accessory protein is CsgA, CsgB, CsgC, CsgD,CsgE, or CsgF.